Skip to main content

Recent advancements in identification and detection of saliva as forensic evidence: a review



Saliva is the most common biological evidence found at any crime scene next to blood. It is a clear liquid which makes it immune to any possible evidence of alteration by the perpetrator. In forensics, saliva is used as biological evidence and is very helpful in determining various aspects of an individual such as sex, individuality, ABO blood groups, microbial signature, biomarkers, or habits like smoking.

Main body

Saliva shares a great resemblance with plasma as it encompasses similar organic or inorganic compound contents. In forensic casework, identifying any evidence is the primary goal to establish the groundwork for further investigation. Saliva may be found in the form of a pool or stained form, but its identification is challenging because of its transparency. It has been widely used as an informative tool in forensic situations like poisoning, hanging, or cases of drug abuse, etc. for more than two decades now. Over the years, many proposed ways or methods have been identified and described, which helped in the detection and identification of saliva as evidence.


This review article represents the significance of saliva as important forensic evidence, along with the different forms it may be encountered at the crime scene. The use of diverse collection and detection methods, over the past few decades, has been discussed. An attempt has been made to collect the available data, highlighting the merit and demerits of different identification techniques. The relevant data has been collected from all the published and reported literature (1987–2021).


Identifying biological evidence obtained from any crime scene is crucial for forensic investigations. These evidences are reliable for linking crime to criminals and in the recreation of the crime scene. Establishing the identity of body fluid or even identifying evidence as body fluid is enough to influence the case’s outcome. The primary body fluids found at any crime scene are blood, semen, saliva, sweat, urine, and vaginal secretions. Each of them has their specific screening methods for identification. Blood is the most common biological fluid present at most crime scenes. In cases like homicide, murder, and sexual assault- blood, saliva, and semen is very common evidence obtained, as compared to others. In case of suicide, saliva and urine are commonly observed biological fluids. These fluids could be present either in a pool form or on any inanimate object that comes in contact with either the victim or assailant.

There is a common tendency of criminals to dispose of the weapon of offense or any other visible evidence like blood stains to avoid getting caught. However, saliva, a transparent liquid has an advantage over blood. Saliva is commonly found in cases such as homicide, sexual assault, burglary, and hanging. In homicide and sexual assault cases, saliva is usually deposited in bite marks. Also, proximity between victim and assailant(s) often leads to the deposition of saliva on each other or surroundings. In case of burglary, sometimes half-eaten fruits or edibles are the sources of salivary evidence, and in case of hanging, the unconscious dripping of saliva is very commonly observed. The evidence which is convenient to send to the laboratory is sent as it is, meanwhile the evidence which is not, is swabbed with sterile swabs and sealed and packed for screening analysis in the laboratory. These screening tests are presumptive, followed by a confirmatory test. The collection of these shreds of evidence is a challenge, too, as many factors influence the quality of evidence (Lee et al. 2001; Acosta et al. 2002; Magalhães et al. 2015).

Salivary gland secretions comprise a gingival crevicular fluid, exfoliated oral epithelial cells, and bacteria that are all combined to form the whole saliva. Saliva is a common fluid for forensic analysis due to the additional benefits of its noninvasive mode of collection, even by individuals with less experience, and the avoidance of intrusion into private functions when collected under direct observation. Saliva aids in many ways such as personal identification by DNA profiling, analysis of drug abuse, animal bite marks, or sex determination from bite marks. Saliva is the only biological fluid that shows similar characteristics to plasma (Saxena and Kumar 2015; John et al. 2018). Salivary fluid is an exocrine secretion that contains 99% water and a variety of electrolytes (sodium, potassium, calcium, chloride, magnesium, bicarbonate, phosphate) as well as proteins that include enzymes, immunoglobulins and other antimicrobial agents, mucosal glycoproteins, traces of albumin, and some polypeptides and oligopeptides that are significant for oral health. Additionally, there is glucose, nitrogenous compounds like urea and ammonia, desquamated epithelial cells, leucocytes, and oral microbes ( De Almeida et al. 2008).

Saliva has many perks and is considered advantageous over blood because unlike blood, its collection is non-invasive, easy, and safer, with a low risk of contamination, especially from transmitted diseases such as hepatitis (Mago et al. 2016; John et al. 2018). Since saliva exists as a clear liquid, it can be often ignored as evidence and is also less prone to any chances of alteration by criminals. It could be present in the pool or the stained form on any surface. Saliva gets deposited on human skin by various means like biting, licking, spitting, and sucking (Schenkels et al. 1995; Sweet et al. 1997; Raj et al. 2018). It can also be assessed from prominent bite marks on food items (De Oliveira Musse et al. 2019) or other inanimate objects such as cigarette butts, straws, etc. (Pawar et al. 2018; Casey et al. 2013). An investigator should always assess the victim for any possible deposition of saliva samples. Since it is difficult to locate definite saliva sites on the body, the significant sites with high recovery chances of saliva are the face, neck, chest, shoulder, abdomen, forearm, and thighs (Jimsha et al. 2020). Apart from these significant body sites, the crime scene is also thoroughly investigated for any inanimate object with susceptibility to the presence of saliva on it. The inanimate objects received as evidence in various types of cases include cloth, personal belongings such as mobiles, cigarette butts, straws, cans, half-eaten edibles, and many more (Sweet and Hildebrand 1999; Pawar et al. 2018).

The implication of saliva on the human body is very different from inanimate objects. The environmental and other external factors such as personal habits like showering or clothing could also hinder the amount of saliva recovered from the skin. Sweet and Shutler 1999, reported a case study, in which the saliva samples were effectively collected from a prominent bite mark present on a female body submerged in water for a period of four to five hours. They generated the DNA profile from bite marks that helped the investigation. Similarly, Chávez-Briones et al. 2015 reported a study on a homicide case where saliva was recovered from bite marks present on the breasts of the victim. They successfully generated a DNA profile from the saliva samples and matched it with that of the suspect.

The present review focused on the different detection and identification techniques or assays used to establish the identity of saliva as crucial forensic evidence during an investigation. It also summarizes the outlook of effective techniques used to identify saliva in the recent past. A comparative assessment in terms of the significance of various techniques has also been discussed. This paper is organized as follows-

  • Collection of saliva samples by using different methods.

  • Various preliminary tests were used in the detection of saliva. This includes polilight, Phadebas, SALIgAE, and immunochromatographic strip tests.

  • Various advanced screening techniques for saliva. This portion includes a wide range of laboratory techniques used for the detection of saliva including DNA methylation, and microscopic methods, among others.

  • Comparison of techniques based on their destructive and non-destructive type.

Main text

Collection of saliva samples

The collection of saliva is a very intricate process that is usually carried out by two swabbing techniques. The choice of technique depends on the type of surface on which evidence gets deposited. In a single swab approach, a wet sterile cotton swab which is moistened by distilled water/normal saline is rolled over the collection site. The swabbing is done without applying extra pressure to avoid the collection of the substrate material. Though, in double swabbing method, the sample site is swabbed twice. First, a wet cotton swab is rolled over the site, followed by a dry swab (Sweet et al. 1997). This method ensures maximum collection of saliva from the substrate. The Double swab technique is followed for absorbent surfaces such as skin, cloth, and half-eaten food items, etc. for non-absorbent surfaces, such as plastic evidence, and glasses, etc. the single swab technique is administered (Hedman et al. 2020). While swabbing, the investigator must be careful not to swab beyond the desired site as it could cause unnecessary contamination leading to improper or mixed DNA profiling. The double swabbing collection method has been preferred over single swabbing since the former ensures obtaining the maximum amount of evidence from the surface.

After collecting the saliva, the evidence is treated with a series of techniques. These techniques are broadly classified into two—destructive and non-destructive. The technique which destroys the integrity of evidence after the assessment is known as the destructive technique. In this the evidence treated, cannot be used for further processing or stored for later. Though, integrity of evidence is not affected after assessment in case of non-destructive techniques.

Preliminary tests

The preliminary tests state that saliva “might” be present in the submitted evidence, followed by confirmatory tests. Each test has its advantages and limitations. These tests consume a significant amount of time as well as evidence. The evidence loss in identification analysis cannot be retrieved to its original state unless the test is non-destructive. Due to this, there is very little evidence left to test for DNA profiling to establish the identity. The preliminary tests, a.k.a presumptive tests, for saliva, are based on the amylase enzyme activity. But this amylase is not solely present in saliva; it is also associated with other body fluid secretions (Wornes et al. 2018). There are two types of amylases, coded by AMY1 (α-amylase-1) and AMY2 (α amylase-2) locus present on chromosome 1. AMY1 type is present in saliva, breast milk, and sweat, while AMY2 is generally present in semen, pancreas, and vaginal fluid respectively (Sensabaugh 1982; Greenfield and Sloan 2002). α amylase-1 is found to be excessive in saliva as compared to other body fluids and can be easily distinguished from α amylase-2 by radial diffusion assay (Quarino et al. 1993).

Polilight detection

Polilight is a portable, high-intensity light source used to locate body fluids. This method is the only non-destructive type of presumptive technique for the detection of saliva (Table 1). It produces an intense narrow band of light with a wavelength between 310 and 650 nm (Vandenberg and Oorschot 2006). Forensic light source (FLS) is a common term adapted for any illuminating light source that aids in forensic investigation. It is also known as an Alternating light source (ALS) (Lennard and Stoilovic 2004). This method helps in visibility and enhances it for photography. This is rapid and less laborious, particularly for large surfaces.

Table 1 Various studies on polilight detection of saliva

Although, saliva is often difficult to locate under Polilight because of the lower fluorescence intensity (Camilleri et al. 2006); also, the absorbency of the surface acts as a hindrance to fluorescence (Vandenberg and Oorschot 2006). There are goggles having certain filters that allow only desirable wavelengths and give peculiar observations (Chuena and Eea 2010) (Table 2). The disadvantage of polilight is that the color of the material hampers the strength of the appearance of the stain (Vandenberg and Oorschot 2006) also, fluorescence patterns are similar to that of other body fluids, leading to non-specificity (Camilleri et al. 2006).

Table 2 Excitation wavelength coupled with suitable goggles

Phadebas test

The Phadebas test is a commonly used presumptive assay. Its active component is a blue dye-bound DSM-P, microsphere. The test relies on the alpha-amylase activity of saliva and is prone to multiple pseudo-positive results. A positive reaction is usually confirmed after the release of blue dye, which results from hydrolyses of starch in the presence of amylase (Hedman et al. 2008). It gives negative results with animal saliva, fruits, vegetables, and some cleaning solutions (Pang and Cheung 2008; Casey and Price 2010; Feia and Novroski 2013); other body fluids (Rao et al. 2020).

In a recent study, the Phadebas press test has also been reported to detect saliva stains on certain fabrics, and aged stained samples (up to 3 months) (Woodford et al. 2021). Some studies have shown its worth compared to other methods such as Polilight (Hedman et al. 2008), starch iodine, and SALIgAE (Myers and Adkins 2008). On the contrary, the inability of the Phadebas test was also reported to presume the identity of saliva (Olsén et al. 2011). It depends upon the type of evidence and the surface on which saliva is present.

SALIgAE test

It is a colorimetric approach to eliminate any possible false-positive reactions given by its counterparts methods- Phadebas and Polilight. Hence, it is a more sensitive approach for preliminary saliva detection (Pang and Cheung 2008; Liang and Roy 2014; Harbison and Fleming 2016). The potency and accuracy of SALIgAE have been studied many times with different approaches. It shows sensitivity, irrelevant to the surface on which the stain is present, like envelopes, soda bottles/cans, and mouth masks, and shows specificity towards human saliva (Miller and Hodges 2005). A positive reaction is the yellow color change of an otherwise colorless solution. However, the reaction time must be confined to 5 min to avoid false-positive by other body fluids, which generally takes more than 5 min (Lim et al. 2008). On comparative analysis of SALIgAE with other assays, it is found to be better and more sensitive than Phadebas.

Apart from these benefits, it possesses some disadvantages because of the colorimetric result (Myers and Adkins 2008). For analysis in samples mixed with blood, the significant disadvantage of this colorimetric assay is the generation of false positive reaction. It is also time-consuming, or the procedure involves maximum dilution of the sample beforehand, leading to disadvantages and abuse of evidence (Silenieks 2006; Myers and Adkins 2008; Park et al. 2015).

Immunochromatographic strip test

A lateral flow immunochromatographic strip works on engaging two monoclonal antibodies present in saliva, making them more sensitive (Casey and Price 2010). A common example of this strip is Rapid Stain Identification (RSID) kit. This test is different and much more sensitive than Phadebas or SALIgAE because of its serological approach instead of colorimetric (Fig. 1). Many studies have compared these three approaches, and RSID emerged to be much more sensitive and time-effective than its counterparts. This qualitative assay generates positive or negative results depending on the presence or absence of a red or blue line on the “Test zone” after 10–15 min of sample addition (Sinelnikov et al. 2013). The accuracy, reproducibility, and sensitivity of RSID to detect saliva on different surfaces (cigarette butts, cans, bottles, etc.) are as low as 1µL (Old et al. 2009). This technique is efficient, portable, and potent to remove any ambiguity if it occurs. Since it detects the presence of alpha-amylase, which is also present in other body fluids such as urine, and vaginal secretion, the sensitivity of RSID with saliva is much greater (Sari et al. 2020).

Fig. 1
figure 1

Sensitivity comparison of Phadebas, SALIgAE, and RSID test

The efficacy of an immunochromatographic strip also depends upon the type of substrate on which the sample has been placed. Saliva has a tendency to get absorbed into porous material or substrates thereby making it more difficult to examine as compared to non-absorbent, non-porous surfaces. Also, environmental conditions are responsible for the degradation of saliva, like- light, heat, air, and humidity (Castelló et al. 2017). Though RSID can detect saliva with up to 10,000-fold dilution and amylase with up to 20,000-fold dilution (Old et al. 2009), these conditions may generate different results. The RSID technique has also been reported on samples degraded by humid soil and showed positive results which otherwise showed negative results by Phadebas (Ohta and Sakurada et al. 2019). This assay also showed efficacy in detecting saliva from expirated blood spatters or blow artifacts. The other assays either failed or showed faint or pseudo-positive, but RSID gave positive results (Silenieks 2006; Park et al. 2015; Thompson et al. 2022).

Advanced screening/detection techniques

Various detection techniques are considered confirmatory to establish the identity of saliva. Some of these tests have been followed for decades, and some are still emerging.

Immunological techniques

Immunology assays are antigen–antibody-based reactions. These assays not only aid in detection but in species determination as well. Enzyme-linked immunosorbent assay (ELISA) was performed using horseradish peroxidase conjugate combined with monoclonal antibodies to detect the alpha-amylase activity in saliva (Komuro et al. 1995). This assay has shown no cross-reactivity with either pancreatic or bacterial amylase, but there were still some false positives- 13% of other body fluid (Quarino et al. 2005). To overcome this, Statherin (STATH), a low molecular weight phosphoprotein secreted by the Parotid gland, was used for detection since it is only present in saliva. The findings gave positive for saliva with no cross-reactivity in ELISA. Also, old-age and mixed saliva samples were readily detected (Akutsu et al. 2010).

Other proteins, known as proline-rich proteins (PRPs), are present in two forms- acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2 (PRB2). They are specific for saliva, especially the PRH1/2 expressed explicitly in salivary glands (Fábián et al. 2012). These PRPs were used in ELISA to detect saliva and to compare the sensitivity, efficiency, and specificity with STATH (Igoh 2015). Similarities in the detection rate and sensitivity of STATH and PRH1/2 followed by PRB2 were observed. However, the specificity towards saliva of PRH1/2 was higher than STATH, stating the former to be of forensic importance.

Other immunological techniques such as immunoelectrophoresis have also been attempted for saliva detection but showed multiple cross-reactivities, making them unreliable for the detection of saliva (Virkler and Lednev 2009).

Microscopy techniques

Microscopic techniques like SEM (Scanning Electron Microscope) coupled with EDX (Energy Dispersive X-Ray) can identify a specific metal concentration for detection. Some of the trace elements like sodium, phosphorus, sulfur, chlorine, potassium, and calcium are also present in saliva at different concentrations. However, potassium is shown to have the most prominent peak in saliva samples and thus can be used for detection purposes (Seta 1977; Dube et al. 2020).

RNA profiling

RNA is unstable and prone to degradation, but many studies have shown its stability in samples and its massive use for forensics (Juusola and Ballantyne 2005; Chong et al. 2015; Park et al. 2006). The availability of RNAseq by Massively parallel sequencing (MPS) has made the rapid discovery and characterization of novel transcripts and other RNA regions such as non-coding, micro, and small RNA, possible (Trapnell et al. 2010; Roewer 2013). MicroRNA is a class of small non-coding RNA (ncRNA) molecules with 18–24 nucleotides that act as essential regulators for multiple cellular processes (Courts and Madea 2011) and are less susceptible to environmental decay (Sirker et al. 2017). The degradation rate of RNA in various biological samples over 6 months shows a similar decay pattern that can be analyzed to estimate the sample’s age (Simard et al. 2012).

The first two saliva-specific RNA genes to be identified were statherine (STATH) and histatin3 (HTN3) ( Ballantyne and Juusola, 2009; Haas et al. 2008; Haas et al. 2009; Chong et al. 2015; Watanabe et al. 2017). Later, Mucin7 (MUC7) gene was also reported along with STATH and HTN3 genes in degraded saliva samples of about two to 6 weeks (Sharma and Vogel 2009). The technique used to analyze these genes are reverse transcription endpoint PCR and RT-PCR (Real-time Polymerase Chain Reaction) (Haas et al. 2009 ). However, Liu et al. (2020) further used capillary electrophoresis (CE)-based method for the examination of these saliva-specific genes. Other than these, five other stable RNA genes—SPRR3, SPRR1A, KRT4, KRT6A, and KRT13—also provided good results for up to 180 days old saliva samples (Zubakov et al. 20082008).

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a new technique used for detection, a one-step amplification of a specific RNA sequence. This method is sensitive and provides high specificity (Tsai et al. 2018; Jackson et al. 2020; Layne et al. 2021).

DNA methylation

DNA, compared to RNA, is a more stable molecule in biological fluids (Marguet and Forterre 1994; Park et al. 2014). Methylation is a genetically programmed type of DNA modification in mammals, occurring at the 5′ position of the cytosine in the CpG dinucleotide sequence (Miranda and Jones 2007). It plays an essential role in the development and differentiation of cells by controlling gene expression through changes in the chromatin structure and tissue-specific patterns (Hashimshony et al. 2003; Straussman et al. 2009; Schilling and Rehli 2007). DNA methylation is an epigenetic modification that can provide important information if explored vividly (Frumkin et al. 2011; Varriale 2014). The DNA methylation markers show similarity in cellular and extracellular DNA, which suggests their positive examination in the absence of cells (Fu et al. 2015). So far, multiple DNA methylation markers for saliva have been reported for forensic purposes (Table 3).

Table 3 DNA methylation markers for saliva

DNA methylation can be done by various techniques, the most common is a chemical modification of cytosine residues by sodium bisulfate (Gomaa et al. 2017). This modification can speed up by using high-concentration bisulfate at high temperatures (Shiraishi and Hayatsu 2004). However, bisulfite conversion can lead to the undesirable side effect of DNA degradation, which is not ideal if the sample concentration is low or poor quality (Lin et al. 2016). Methylation-sensitive restriction enzyme PCR (MSRE-PCR), an alternate strategy, was introduced to overcome the issue, but it worked only for semen identification (Lin et al. 2016; Wasserstrom et al. 2013). Methylation SnaPshot is the second most used technique; it has the advantage of simultaneous analysis by constructing multiplex methylation SnaPshot. Another introduction to this is multiplex Snapshot microarray which combines different markers. Bisulfite genomic sequencing is a fundamental gold-standard methylation technique because it provides a qualitative, quantitative, and efficient approach to identifying 5-methylcytosine at single base-pair resolution (Gomaa et al. 2017). Next Gen Sequencing has been suggested as an aid to overcome any possible false positives (Gauthier et al. 2019).

Spectroscopic technique

The advancement and recent developments in body fluid analysis using vibrational spectroscopy have shown excellent potential for an alternative approach (Elkins 2011; Virkler and Lednev 2008). This technique is universal, non-destructive, and label-free (Vyas et al. 2020).

UV–Visible spectroscopy

UV–Visible spectroscopy has been improvised over the years, with many advancements and changes from a short range to a broader range (Fiedler et al. 2008; Zapata et al. 2015a). Multiple studies have been done to show the specific wavelength range of different body fluids. However, the similar response of one body fluid to another fluid’s characteristic spectra, questions the technique’s specificity. This technique is prone to multiple false positives as other substances may hinder results (Zapata et al. 2015b).

Fluorescent spectroscopy

Fluorescent spectroscopy uses tryptophan, an endogenous fluorophore in alpha-amylase, as a prevalent probe in dried saliva stains on human skin. It shows a characteristic emission spectrum at 345–355 nm (Soukos et al. 2000; Nanda et al. 2011; Sikirzhytski et al. 2011).

Raman spectroscopy

Raman is a widely used and accepted technique in body fluid identification because it provides better spatial resolution and doesn’t react with water (Sikirzhytski et al. 2012; Virkler and Lednev 2010). Apart from that, Phenylalanine, present in amylase and lipase, gives peculiar peaks of saliva in Raman and Thiocyanate, which is found in the saliva of a smoker (Virkler and Lednev 2008). The saliva spectra are high compared to other body fluids and can be discriminated against when mixed with blood or semen (Quarino et al. 2005; Hardy et al. 2022). Compared to other amino acids, the spectra of tryptophan are single maximum. This assay can also be used to determine the sex of the sample (Muro et al 2016).

Fourier transform infrared spectroscopy

Another robust technique is Fourier transform infrared spectroscopy (FTIR), which can detect multiple spectra of saliva based on alpha-amylase and lysozymes (Orphanou et al. 2015; Takamura et al. 2018). However, a disadvantage of vibrational spectroscopy is the multiple spectra of a single body fluid. Since a body fluid is a complex mixture, it gives inhomogeneous complex spatial distributions. Moreover, environmental degradation of samples hinders results due to multiple contaminations (Orphanou et al. 2015).

Microbial detection for saliva identification

Healthy saliva is a mixture of proteins, carbohydrates, antibacterial proteins, white blood cells, and many other components. Though it has antibacterial immunoglobulins and WBCs, forming the first line of defense, it does nurture an array of microbial flora (Dash and Das 2018). On average, the total microscopic count is approximately 750 million oral bacteria cells per milliliter of saliva, and of these, streptococci are the most abundant. The presence of these bacteria solely in the saliva has been studied over the years by various techniques such as PCR (Nakanishi et al. 2009; Ali et al. 2013; Choi et al. 2014) and loop-mediated isothermal amplification (Nakanishi et al. 2011). Over the years, the interest in microbiome identification of body fluids has increased drastically among forensic scientists. There have been multiple studies that show definite positive detection but are performed differently (Fig. 2).

Fig. 2
figure 2

Flowchart showing the different types of Microbes in saliva

Streptococcus salivarius is one of the most common bacteria, followed by Streptococcus mutans, responsible for dental caries (Ali et al. 2013). Streptococcus salivarius (a prominent member of oral microbiota), Streptococcus sanguinis (most abundant species in oral biofilms), and Neisseria subflava (normal flora of the oral cavity and respiratory tract) are the most common microbes used to identify saliva by different methods combined with RT PCR such as OB mRT-PCR (Jung et al. 2018) and colorimetric detection of bacterial DNA amplicons on an immunochromatographic strip (ICS) (Lee et al. 2018).

These techniques are novel, more convenient, user-friendly, and cost-effective. The cross-examination of samples by conventional methods generates a result that favors microbial detection and is specific for body fluid and species. The sensitivity of this method is high in mixed samples as well (Li et al. 2019).

In degraded samples, the identification of saliva using microbial profiling has shown high sensitivity compared to other techniques. Saliva in samples degraded up to 30 days has been analyzed by examining the 16S rRNA V4-V5 region and successfully amplifying only 92% of total saliva samples (Fig. 3) (Dobay et al. 2019).

Fig. 3
figure 3

Microbial community in saliva

Another study reported the efficacy of oral gram-positive and gram-negative bacteria for DNA-based identification of highly degraded salivary samples. It was observed that oral gram-positive bacteria served as a better source for DNA-based identification as the same could be detected successfully in environmentally exposed and UV-irradiated samples (Table 4) (Ohta and Sakurada et al. 2019).

Table 4 Efficacy of oral gram-positive and gram-negative bacteria for DNA-based identification

The latest advancement in this technique is developing a novel smartphone-based bacteria sensor that assesses two oral bacteria—S. salivarius and S. sanguinis (Li 2020). The test strip gives dose-sensitive color (blue-emitting silicon carbide quantum dots and red-emitting gold nanoclusters) when introduced under a 365-nm UV lamp. The color intensity is directly proportional to the concentration of bacteria in the sample. This method can identify two bacteria simultaneously in 20 min, unlike other oral bacteria detection methods, and is the latest advancement in the forensic microbiome.

Destructive and non-destructive assessment of saliva samples

The above-mentioned assessment techniques for saliva can be further classified into destructive and non-destructive categories. Destructive methods often result in the consumption of samples in a way that the samples cannot be used for further processing for DNA analysis. An overview of various destructive and non-destructive techniques used in the examination of saliva samples has been presented in Table 5.

Table 5 List of destructive techniques used in saliva analysis


Saliva holds much relevance in forensics as any other body fluid. Its non-invasive collection, wide benefit, and colorless appearance make it good evidence. The detection process of saliva offers a destructive and non-destructive assessment. The detection process provides information on the quality and quantity of saliva present on the substrate. After establishing the identity of saliva, it becomes a major source of DNA, which is further processed for DNA profiling.

The preliminary detection is based on the presence of α- amylase, saliva-specific antibodies, and the fluorescence activity of saliva. The polilight-based detection is portable and low cost but shows specific colored light that is impossible to read on bright backgrounds. The light emitted by saliva mimics the fluorescence of other body fluids, which could lead to either false identification or more time consumption until other techniques verify the identity. The Phadebas and SALIgAE tests rely on the presence of α-amylase present on the test saliva samples. These tests are simple to use but have a disadvantage of cross-reactivity, time efficacy, and wastage of samples. Immunochromatographic kit-based detection of monoclonal antibodies provides better sensitivity than the former three, but tends to give false negative results with other body fluids.

In advanced screening, the detection of saliva must be established for further downstream processing of evidence. Each technique is unique but possesses some disadvantages such as the RNA being highly unstable, and its profiling for identification can be costly and time-consuming. The DNA methylation technique can identify body fluid and individuals with single processing. Among all these, the NextGen sequencing technique, so far, is a much better approach but NGS is costly and cannot be available everywhere.

On the other hand, the spectroscopy techniques are robust, non-destructive, and possess considerable merits. The only demerit is its multiple spectra of a single body fluid, which could worsen if the sample is contaminated or mixed. These techniques maintain the integrity of the sample and produce more satisfactory results. Microbial profiling is the new advancement and interest for detection, falling under forensic microbiology. However, this technique requires skills and is expensive. The survival rate of microbes in the environment is much more than any nucleic acid or protein. It can be concluded that despite having an ample amount of techniques for identification and detection, there is still a lack of a definitive technique for saliva examination. The technique which is robust, feasible, cost-effective, and does not compromise the integrity of the exhibit still needs to be explored.

Availability of data and materials

Not applicable.



Deoxyribonucleic acid


Forensic light source


Alternate light source


α-Amylase 1/ α-amylase 2


Rapid stain identification series


Enzyme-linked immunosorbent assay




Proline-rich proteins


Scanning electron microscope


Energy dispersive X-ray


Ribonucleic acid


Massive parallel sequencing




Quantitative polymerase chain reaction


Reverse transcription–loop-mediated isothermal amplification


Methylation-sensitive restriction enzymes–polymerase chain reaction




Fourier transform infrared


White blood cells


Real-time polymerase chain reaction


  • Acosta ML (2002) Collecting evidence for domestic and sexual assault: highlighting violence against women in health care system interventions. Int J Gynecol Obstet 78:S99–S104

    Article  Google Scholar 

  • Akutsu T, Watanabe K, Fujinami Y, Sakurada K (2010) Applicability of ELISA detection of statherin for forensic identification of saliva. Int J Legal Med 124(5):493–498

    Article  PubMed  Google Scholar 

  • Ali MM, Shokry DA, Zaghloul HS, Rashed LA, Nada MG (2013) PCR applications in identification of saliva samples exposed to different conditions (streptococci detection based). Pakistan J Biol Sci 16(12):575–579

    Article  CAS  Google Scholar 

  • Ballantyne J, Juusola J (2009) Messenger RNA Profiling: Body Fluid ID using Multiplex Reverse Transcription-polymerase chain reaction (RT-PCR)[DIV]

    Google Scholar 

  • Beyer V, Schwender K, Glaub A, Johann KS, Schürenkamp M, EUROFORGEN-NoE Consortium (2017) Independent validation of body fluid-specific CpG markers and construction of a robust multiplex assay. Forensic Science International: Genetics 29:261–268

    Article  PubMed  Google Scholar 

  • Camilleri E, Silenieks E, Henry J (2006) Locating Saliva Stains using the Polilight® and SALIgAE® Sprayy. Forensic Science SA, Government of South Australia, Evidence Recovery and Biology Analytical Groups

    Google Scholar 

  • Carter-Snell C, Soltys K (2005) Forensic ultraviolet lights in clinical practice: evidence for the evidence. Can J Police Secur Serv 3(2):79–85

    Google Scholar 

  • Casey DG, Price J (2010) The sensitivity and specificity of the RSID™-saliva kit for the detection of human salivary amylase in the Forensic Science Laboratory, Dublin Ireland. Forensic Sci Int 194(1–3):67–71 (Casey and Price 2010)

    Article  CAS  PubMed  Google Scholar 

  • Casey L, Engen S, Frank G (2013) Quantitative analysis of the DNA distribution on cigarette butt filter paper. J Forensic Sci 58(2):470–473

    Article  CAS  PubMed  Google Scholar 

  • Castelló A, Francès F, Verdú F (2017) The effectiveness of the RSID confirmatory test kit for human alpha amylase: the effects of environmental factors and substrate materials. Aust J Forensic Sci 49(2):217–224.

  • Chávez-Briones ML, Hernández-Cortés R, Jaramillo-Rangel G, Ortega-Martínez M (2015) Relevance of sampling and DNA extraction techniques for the analysis of salivary evidence from bite marks: a case report. Genet Mol Res 14(3):10165–10171

    Article  PubMed  Google Scholar 

  • Choi A, Shin KJ, Yang WI, Lee HY (2014) Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA. Int J Legal Med 128(1):33–41

    Article  PubMed  Google Scholar 

  • Chong KWY, Wong Y, Ng BK, Thong Z, Syn CKC (2015) Development of a RNA profiling assay for biological tissue and body fluid identification. Forensic Sci Int Genet Suppl Ser 5:e196–e198

    Article  Google Scholar 

  • Chuena LW, Eea KB (2010) Forensic light sources for detection of biological evidences in crime scene investigation: a review. Malays J Forensic Sci 1:17–28

    Google Scholar 

  • Courts C, Madea B (2011) Specific micro-RNA signatures for the detection of saliva and blood in forensic body-fluid identification. J Forensic Sci 56(6):1464–1470

    Article  CAS  PubMed  Google Scholar 

  • Dash HR, Das S (2018) Microbial degradation of forensic samples of biological origin: potential threat to human DNA typing. Mol Biotechnol 60(2):141–153

    Article  CAS  PubMed  Google Scholar 

  • De Almeida PDV, Gregio AM, Machado MA, De Lima AA, Azevedo LR (2008) Saliva composition and functions: a comprehensive review. J Contemp Dent Pract 9(3):72–80

    Article  PubMed  Google Scholar 

  • De Oliveira Musse J, Marques JAM, Remualdo V, Pitlovanciv AK, da Silva CAL, Corte-Real F, Corte-Real AT (2019) Deoxyribonucleic acid extraction and quantification from human saliva deposited on foods with bitemarks. J Contemp Dental Pract. 20(5):549

    Google Scholar 

  • Dobay A, Haas C, Fucile G, Downey N, Morrison HG, Kratzer A, Arora N (2019) Microbiome-based body fluid identification of samples exposed to indoor conditions. Forensic Sci Int Genet 40:105–113

    Article  CAS  PubMed  Google Scholar 

  • Dube S, Tharmavaram M, Pandey G, Rawtani D, Mustansar Hussain C (2020) Sensors for the Detection of Biological Fluids. In Technology in Forensic Science pp. 239–258. Wiley.

  • Elkins KM (2011) Rapid presumptive “fingerprinting” of body fluids and materials by ATR FT-IR spectroscopy. J Forensic Sci 56(6):1580–1587

    Article  CAS  PubMed  Google Scholar 

  • Fábián TK, Hermann P, Beck A, Fejérdy P, Fábián G (2012) Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 13(4):4295–4320

    Article  PubMed  PubMed Central  Google Scholar 

  • Feia A, Novroski N (2013) The evaluation of possible false positives with detergents when performing amylase serological testing on clothing. Journal of forensic sciences 58:S183–S185 (Feia and Novroski 2013)

    Article  CAS  PubMed  Google Scholar 

  • Fiedler A, Rehdorf J, Hilbers F, Johrdan L, Stribl C, Benecke M (2008) Detection of semen (human and boar) and saliva on fabrics by a very high powered UV-/VIS-light source. Open Forensic Sci J 1(1):12–15.

  • Forat S, Huettel B, Reinhardt R, Fimmers R, Haidl G, Denschlag D, Olek K (2016) Methylation markers for the identification of body fluids and tissues from forensic trace evidence. PLoS ONE 11(2):e0147973

    Article  PubMed  PubMed Central  Google Scholar 

  • Frumkin D, Wasserstrom A, Budowle B, Davidson A (2011) DNA methylation-based forensic tissue identification. Forensic Sci Int Genet 5(5):517–524

    Article  CAS  PubMed  Google Scholar 

  • Fu XD, Wu J, Wang J, Huang Y, Hou YP, Yan J (2015) Identification of body fluid using tissue-specific DNA methylation markers. Forensic Sci Int Genet Suppl Ser 5:e151–e153

    Article  Google Scholar 

  • Gauthier QT, Cho S, Carmel JH, McCord BR (2019) Development of a body fluid identification multiplex via DNA methylation analysis. Electrophoresis 40(18–19):2565–2574

    CAS  PubMed  Google Scholar 

  • Ghai M, Naidoo N, Evans DL, Kader F (2020) Identification of novel semen and saliva specific methylation markers and its potential application in forensic analysis. Forensic Sci Int Genet 49:102392

    Article  CAS  PubMed  Google Scholar 

  • Gomaa R, Salehi J, Behl S (2017) DNA methylation as a biomarker for body fluid identification. Arab J Forensic Sci Forensic Med 1:681–694

    Article  Google Scholar 

  • Greenfield A, Sloan MM (2002) Identification of biological fluids and stains. In Forensic Science (pp. 231–248). CRC Press. (Greenfield and Sloan 2002)

  • Haas C, Klesser B, Kratzer A, Bär W (2008) mRNA profiling for body fluid identification. Forensic Sci Int Genet Suppl Ser 1(1):37–38

    Article  Google Scholar 

  • Haas C, Klesser B, Maake C, Bär W, Kratzer A. (2009). mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Intern Genet 3(2):80–88.

  • Harbison S, Fleming R (2016) Forensic body fluid identification: state of the art. Res Rep Forensic Med Sci 6(2016):11–23

    Google Scholar 

  • Hardy M, Kelleher L, de Carvalho Gomes P, Buchan E, Chu HOM, Goldberg Oppenheimer P (2022) Methods in Raman spectroscopy for saliva studies–a review. Appl Spectrosc Rev 57(3):177–233

    Article  Google Scholar 

  • Hashimshony T, Zhang J, Keshet I, Bustin M, Cedar H (2003) The role of DNA methylation in setting up chromatin structure during development. Nat Genet 34(2):187–192

    Article  CAS  PubMed  Google Scholar 

  • Hedman J, Gustavsson K, Ansell R (2008) Using the new Phadebas® Forensic Press test to find crime scene saliva stains suitable for DNA analysis. Forensic Sci Int Genet Suppl Ser. 1(1):430–432 ((Hedman et al. 2008))

    Article  Google Scholar 

  • Hedman J, Jansson L, Akel Y, Wallmark N, Liljestrand RG, Forsberg C, Ansell R (2020) The double-swab technique versus single swabs for human DNA recovery from various surfaces. Forensic Sci Int Genet 46:102253

    Article  CAS  PubMed  Google Scholar 

  • Igoh A, Tomotake S, Doi Y (2015) Detection of proline-rich proteins for the identification of saliva by enzyme-linked mmunosorbent assay. Leg Med 17(3):210–213

    Article  CAS  Google Scholar 

  • Jackson KR, Layne T, Dent DA, Tsuei A, Li J, Haverstick DM, Landers JP (2020) A novel loop-mediated isothermal amplification method for identification of four body fluids with smartphone detection. Forensic Sci Int Genet 45:102195

    Article  CAS  PubMed  Google Scholar 

  • Jimsha VK, Shakila R, Jonathan DM, Ramesh V, Marak F (2020) Bite mark analysis: A case report. J Indian Acad Forensic Med 42(1):66–69

    Article  Google Scholar 

  • John SJ, Rajaji D, Jaleel D, Mohan A, Kadar N, Venugopal V (2018) Application of Saliva in Forensics. Oral Maxillofacial Pathol J 9(2):85–87.

  • Jung JY, Yoon HK, An S, Lee JW, Ahn ER, Kim YJ, Lim SK (2018) Rapid oral bacteria detection based on real-time PCR for the forensic identification of saliva. Scientific Rep 8(1):1–10

    Google Scholar 

  • Juusola J, Ballantyne J (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int 152(1):1–12

    Article  CAS  PubMed  Google Scholar 

  • Karchewski L, Armstrong G, Nicholson ML, Wilkinson D (2014) Assessment of the Leeds Spectral Vision system for detecting biological stains on fabrics. Canad Soc Forensic Sci J 47(4):230–243

    Article  Google Scholar 

  • Komuro T, Mukoyama R, Mukoyama H (1995) Application of enzyme-linked mmunosorbent assay (ELISA) to the medico-legal identification. Nihon rinsho. Japanese J Clin Med. 53(9):2322–2329

    CAS  Google Scholar 

  • Layne T, Jackson K, Scott A, Tanner NA, Piland A, Haverstick DM, Landers JP (2021) Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification. J Forensic Sci 66(3):1033–1041

    Article  CAS  PubMed  Google Scholar 

  • Lee HC, Ladd C (2001) Preservation and collection of biological evidence. Croat Med J 42(3):225–228

    CAS  PubMed  Google Scholar 

  • Lee HY, An JH, Jung SE, Oh YN, Lee EY, Choi A, Shin KJ (2015) Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers. Forensic Sci Int Genet. 17:17–24

    Article  CAS  PubMed  Google Scholar 

  • Lee JW, Jung JY, Lim SK (2018) Simple and rapid identification of saliva by detection of oral streptococci using direct polymerase chain reaction combined with an immunochromatographic strip. Forensic Sci Int Genet 33:155–160

    Article  CAS  PubMed  Google Scholar 

  • Lee WC, Khoo BE, bin Abdullah AL (2012) A simple, low-cost, and portable LED-based multi-wavelength light source for forensic application. In LED and Display Technologies II (Vol. 8560, p. 856005). SPIE.

  • Lennard C & Stoilovic M (2004) Application of forensic light sources at the crime scene. In The practice of crime scene investigation (1st ed) pp. 129–156. CRC Press, Boca Raton.

  • Li X, Ding Y, Ling J, Yao W, Zha L, Li N, Cai J (2019) Bacteria-targeting BSA-stabilized SiC nanoparticles as a fluorescent nanoprobe for forensic identification of saliva. Microchimica Acta 186(12):1–10

    Article  Google Scholar 

  • Li X, Li J, Ling J, Wang C, Ding Y, Chang Y, Cai J (2020) A smartphone-based bacteria sensor for rapid and portable identification of forensic saliva sample. Sensors Actuators B Chem 320:128303.

  • Liang T, Roy R (2014) Ultraviolet-visible spectrophotometry (UV-VIS) and SALIgAE® qualitative and semi-quantitative tools for the analysis of salivary amylase. J Forensic Res 5(6):1

    Google Scholar 

  • Lim SK, Kwak KD, Choi DH, Han MS (2008) Validation of new saliva test using SALIgAE®. Anal Sci Technol 21(1):48–52

    Google Scholar 

  • Lin YC, Tsai LC, Lee JCI, Su CW, Tzen JTC, Linacre A, Hsieh HM (2016) Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system. Forensic Sci Int Genet 25:157–165

    Article  CAS  PubMed  Google Scholar 

  • Liu B, Yang Q, Meng H, Shao C, Jiang J, Xu H, Xie J (2020) Development of a multiplex system for the identification of forensically relevant body fluids. Forensic Science International: Genetics 47:102312

    Article  CAS  PubMed  Google Scholar 

  • Madi T, Balamurugan K, Bombardi R, Duncan G, McCord B (2012) The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis 33(12):1736–1745

    Article  CAS  PubMed  Google Scholar 

  • Magalhães T, Dinis-Oliveira RJ, Silva B, Corte-Real F, Nuno Vieira D (2015) Biological evidence management for DNA analysis in cases of sexual assault. Sci World J 2015:365674.

  • Mago J, Sidhu L, Kaur R, Anurag T (2016) Saliva in forensics. World J Pharm Med Res 2(4):196–198

    Google Scholar 

  • Marguet E, Forterre P (1994) DNA stability at temperatures typical for hyperthermophiles. Nucleic Acids Res 22(9):1681–1686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miller DW, Hodges JC (2005) Validation of Abacus SALIgAE® test for the forensic identification of saliva. WV State police Forensic laboratory, WV

    Google Scholar 

  • Miranda TB, Jones PA (2007) DNA methylation: the nuts and bolts of repression. J Cell Physiol 213(2):384–390

    Article  CAS  PubMed  Google Scholar 

  • Muro CK, de Souza Fernandes L, Lednev IK (2016) Sex determination based on Raman spectroscopy of saliva traces for forensic purposes. Anal Chem 88(24):12489–12493

    Article  CAS  PubMed  Google Scholar 

  • Myers JR, Adkins WK (2008) Comparison of modern techniques for saliva screening. J Forensic Sci 53(4):862–867

    Article  CAS  PubMed  Google Scholar 

  • Nakanishi H, Kido A, Ohmori T, Takada A, Hara M, Adachi N, Saito K (2009) A novel method for the identification of saliva by detecting oral streptococci using PCR. Forensic Sci Int 183(1–3):20–23

    Article  CAS  PubMed  Google Scholar 

  • Nakanishi H, Ohmori T, Hara M, Takada A, Shojo H, Adachi N, Saito K (2011) A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification. J Forensic Sci 56:S158–S161

    Article  CAS  PubMed  Google Scholar 

  • Nanda KDS, Ranganathan K, Umadevi KM, Joshua E (2011) A rapid and noninvasive method to detect dried saliva stains from human skin using fluorescent spectroscopy. J Oral Maxillofacial Pathol JOMFP 15(1):22

    Article  Google Scholar 

  • Ohta J, Sakurada K (2019) Oral gram-positive bacterial DNA-based identification of saliva from highly degraded samples. Forensic Sci Int Genet 42:103–112

    Article  CAS  PubMed  Google Scholar 

  • Old JB, Schweers BA, Boonlayangoor PW, Reich KA (2009) Developmental validation of RSID™-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva. J Forensic Sci 54(4):866–873

    Article  CAS  PubMed  Google Scholar 

  • Olsén EL, Edenberger E, Mattsson M, Ansell R (2011) Phadebas® Forensic Press test and the presence of amylases in body fluids naturally deposited on textile. Forensic Sci Int Genet Suppl Ser 3(1):e155–e156

    Article  Google Scholar 

  • Orphanou CM (2015) The detection and discrimination of human body fluids using ATR FT-IR spectroscopy. Forensic Sci Int 252:e10–e16

    Article  CAS  PubMed  Google Scholar 

  • Pang BC, Cheung BK (2008) Applicability of two commercially available kits for forensic identification of saliva stains. J Forensic Sci 53(5):1117–1122 ((Pang and Cheung 2008))

    Article  PubMed  Google Scholar 

  • Park NJ, Li Y, Yu T, Brinkman BM, Wong DT (2006) Characterization of RNA in saliva. Clin Chem 52(6):988–994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park JL, Kwon OH, Kim JH, Yoo HS, Lee HC, Woo KM, Kim YS (2014) Identification of body fluid-specific DNA methylation markers for use in forensic science. Forensic Sci Int Genet. 13:147–153

    Article  CAS  PubMed  Google Scholar 

  • Park HY, Son BN, Seo YI, Lim SK (2015) Comparison of four saliva detection methods to identify expectorated blood spatter. J Forensic Sci 60(6):1571–1576 ((Park et al. 2015))

    Article  CAS  PubMed  Google Scholar 

  • Pawar SG, Harel VS, Mahajan KD, More BP, Kulkarni KV (2018) Only Cigarette Butt is Left, DNA Fingerprinting Traps the Theft. J Forensic Sci Criminol 6(1):103

    Google Scholar 

  • Quarino L, Hess J, Shenouda M, Ristenbatt RR, Gold J, Shaler RC (1993) Differentiation of alpha-amylase from various sources: an approach using selective inhibitors. J Forensic Sci Soc 33(2):87–94

    Article  CAS  PubMed  Google Scholar 

  • Quarino L, Dang Q, Hartmann J, Moynihan N (2005) An ELISA method for the identification of salivary amylase. J Forensic Sci 50(4):JFS2004417-4

    Article  Google Scholar 

  • Rao A, Rana M, Pradhan A, Sinha M (2020) RNA-and DNA-Based Identification of Body Fluids. Forensic DNA Typing: Principles, Applications and Advancements. Springer, Singapore, pp 87–104

    Chapter  Google Scholar 

  • Roewer L (2013) DNA fingerprinting in forensics: past, present, future. Investigative Genet 4(1):1–10

    Article  Google Scholar 

  • Raj CK, Garlapati K, Karunakar P, Badam R, Soni P, Lavanya R (2018). Saliva as Forensic Evidence using Fluorescent Spectroscopy: A Pilot Study. J Clin Diagn Res 12(9):27–29.

  • Sari D, Hitchcock C, Collins S, Cochrane C, Bruce D (2020) Amylase testing on intimate samples from pre-pubescent, post-pubescent and post-menopausal females: Implications for forensic casework in sexual assault allegations. Aust J Forensic Sci 52(6):618–625

    Article  Google Scholar 

  • Saxena S, Kumar S (2015) Saliva in forensic odontology: A comprehensive update. J Oral Maxillofacial Pathol 19(2):263 ((Saxena and Kumar 2015))

    Article  Google Scholar 

  • Schenkels LC, Veerman EC, Nieuw Amerongen AV (1995) Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med 6(2):161–175.

  • Schilling E, Rehli M (2007) Global, comparative analysis of tissue-specific promoter CpG methylation. Genomics 90(3):314–323

    Article  CAS  PubMed  Google Scholar 

  • Seidl S, Hausmann R, Betz P (2008) Comparison of laser and mercury-arc lamp for the detection of body fluids on different substrates. Int J Legal Med 122(3):241–244

    Article  CAS  PubMed  Google Scholar 

  • Sensabaugh GF (1982) Isozymes in forensic science. Isozymes 6:247–282 ((Sensabaugh 1982))

    CAS  PubMed  Google Scholar 

  • Seta S (1977) Application of scanning electron microscopy and energy dispersive x-ray microanalysis to the criminal identification of body fluid stains. Int Crim Police Rev 307:119–123

    CAS  Google Scholar 

  • Sharma CM, Vogel J (2009) Experimental approaches for the discovery and characterization of regulatory small RNA. Curr Opin Microbiol 12(5):536–546

    Article  CAS  PubMed  Google Scholar 

  • Shiraishi M, Hayatsu H (2004) High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation. DNA Res 11(6):409–415

    Article  CAS  PubMed  Google Scholar 

  • Sikirzhytski V, Sikirzhytskaya A, Lednev IK (2011) Multidimensional Raman spectroscopic signatures as a tool for forensic identification of body fluid traces: a review. Appl Spectrosc 65(11):1223–1232

    Article  CAS  PubMed  Google Scholar 

  • Sikirzhytski V, Sikirzhytskaya A, Lednev IK (2012) Advanced statistical analysis of Raman spectroscopic data for the identification of body fluid traces: semen and blood mixtures. Forensic Sci Int 222(1–3):259–265

    Article  CAS  PubMed  Google Scholar 

  • Silenieks E (2006) The detection of salivary amylase in expirated blood patterns. IABPA Newsletter 22:5–9

    Google Scholar 

  • Silva DS, Antunes J, Balamurugan K, Duncan G, Alho CS, McCord B (2016) Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples. Forensic Sci Int Genet 23:55–63

    Article  CAS  PubMed  Google Scholar 

  • Simard AM, DesGroseillers L, Sarafian V (2012) Assessment of RNA stability for age determination of body fluid stains. Can Soc Forensic Sci J 45(4):179–194

    Article  CAS  Google Scholar 

  • Sinelnikov A, Kalinina A, Old JB, Boonlayangoor PW, Reich KA (2013) Evaluation of rapid stain identification (RSID™) reader system for analysis and documentation of RSID™ tests. Appl Sci 3(3):624–635

    Article  Google Scholar 

  • Sirker M, Fimmers R, Schneider PM, Gomes I (2017) Evaluating the forensic application of 19 target microRNAs as biomarkers in body fluid and tissue identification. Forensic Sci Int Genet 27:41–49

    Article  CAS  PubMed  Google Scholar 

  • Soukos NS, Crowley K, Bamberg MP, Gillies R, Doukas AG, Evans R, Kollias N (2000) A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy. Forensic Sci Int 114(3):133–138

    Article  CAS  PubMed  Google Scholar 

  • Straussman R, Nejman D, Roberts D, Steinfeld I, Blum B, Benvenisty N, Cedar H (2009) Developmental programming of CpG island methylation profiles in the human genome. Nat Struct Mol Biol 16(5):564–571

    Article  CAS  PubMed  Google Scholar 

  • Sweet D, Hildebrand D (1999) Saliva from cheese bite yields DNA profile of burglar: a case report. Int J Legal Med 112(3):201–203

    Article  CAS  PubMed  Google Scholar 

  • Sweet D, Shutler GG (1999) Analysis of salivary DNA evidence from a bite mark on a body submerged in water. J Forensic Sci 44(5):1069–1072

    Article  CAS  PubMed  Google Scholar 

  • Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E (1997) An improved method to recover saliva from human skin: the double swab technique. J Forensic Sci 42(2):320–322

    Article  CAS  PubMed  Google Scholar 

  • Takamura A, Watanabe K, Akutsu T, Ozawa T (2018) Soft and robust identification of body fluid using Fourier transform infrared spectroscopy and chemometric strategies for forensic analysis. Sci Rep 8(1):1–10

    Article  CAS  Google Scholar 

  • Tay JW, Joudo J, Tran T, Ta H, Botting JL, Liew YC, Rye MS (2021) Comparison of Crime-lite® 82S, Polilight® PL400 and Polilight® PL500 for the detection of semen and saliva stains. Aust J Forensic Sci 53(4):483–493

    Article  Google Scholar 

  • Thompson C, Bennett R, Krosch MN, Chaseling J, Wright K (2022) Evaluation of the RSIDTM-Saliva test to detect saliva in expirated bloodstains and development of an ‘in-scene’protocol. Aust J Forensic Sci 54(4):438–449

    Article  Google Scholar 

  • Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnol 28(5):511–515

    Article  CAS  Google Scholar 

  • Tsai LC, Su CW, Lee JCI, Lu YS, Chen HC, Lin YC, Hsieh HM (2018) The detection and identification of saliva in forensic samples by RT-LAMP. Forensic Sci Med Pathol 14(4):469–477

    Article  CAS  PubMed  Google Scholar 

  • Vandenberg N, van Oorschot RA (2006) The use of Polilight® in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests. J Forensic Sci 51(2):361–370

    Article  CAS  PubMed  Google Scholar 

  • Varriale A (2014) DNA methylation, epigenetics, and evolution in vertebrates: facts and challenges. Int J Evol Biol 2014:1–7.

  • Virkler K, Lednev IK (2008) Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids. Forensic Sci Int 181(1–3):e1–e5

    Article  PubMed  Google Scholar 

  • Virkler K, Lednev IK (2009) Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 188(1–3):1–17

    Article  CAS  PubMed  Google Scholar 

  • Virkler K, Lednev IK (2010) Forensic body fluid identification: the Raman spectroscopic signature of saliva. Analyst 135(3):512–517

    Article  CAS  PubMed  Google Scholar 

  • Vyas B, Halamkova L, Lednev IK (2020) A universal test for the forensic identification of all main body fluids including urine. Forensic Chemistry 20:100247

    Article  CAS  Google Scholar 

  • Wasserstrom A, Frumkin D, Davidson A, Shpitzen M, Herman Y, Gafny R (2013) Demonstration of DSI-semen—a novel DNA methylation-based forensic semen identification assay. Forensic Sci Int Genet 7(1):136–142

    Article  CAS  PubMed  Google Scholar 

  • Watanabe K, Akutsu T, Takamura A, Sakurada K (2017) Practical evaluation of an RNA-based saliva identification method. Sci Justice 57(6):404–408

    Article  PubMed  Google Scholar 

  • Wawryk J, Odell M (2005) Fluorescent identification of biological and other stains on skin by the use of alternative light sources. J Clin Forensic Med 12(6):296–301

    Article  PubMed  Google Scholar 

  • Woodford H, Mitchell N, Henry J (2021) Comparative performance of the Phadebas® Forensic Press Test at room temperature and 37° C for the detection of saliva stains on fabric exhibits. Sci Justice 61(2):170–174

    Article  PubMed  Google Scholar 

  • Wornes DJ, Speers SJ, Murakami JA (2018) The evaluation and validation of Phadebas® paper as a presumptive screening tool for saliva on forensic exhibits. Forensic Sci Int 288:81–88

    Article  CAS  PubMed  Google Scholar 

  • Zapata F, Gregório I, García-Ruiz C (2015 b) Body fluids and spectroscopic techniques in forensics: a perfect match. J Forensic Med 1(1):1–7

  • Zapata Arráez F, Fernández de la Ossa MDLÁ, García Ruiz C (2015 a) Emerging spectrometric techniques for the forensic analysis of body fluids. TrAc- Trends in Anal Chem 64:53–63.

  • Zubakov D, Hanekamp E, Kokshoorn M, van Ijcken W, Kayser M (2008) Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med 122(2):135–142

    Article  PubMed  Google Scholar 

Download references


Not applicable


Not applicable.

Author information

Authors and Affiliations



Study conception and design: MU, PS, BJ. Drafting of the manuscript: MU, PS, BJ. Critical revision: MU, PS, KV, BJ. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Bhawana Joshi.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Given by all the authors.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Upadhyay, M., Shrivastava, P., Verma, K. et al. Recent advancements in identification and detection of saliva as forensic evidence: a review. Egypt J Forensic Sci 13, 17 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Saliva
  • Forensic evidence
  • Crime scene
  • Body fluid
  • Identification