|Year : 2022 | Volume
| Issue : 3 | Page : 81-87
Forensic identification of four Indian snake species using single multiplex polymerase chain reaction
Ishani Mitra1, Soma Roy1, Ikramul Haque2
1 Division of Biology, Central Forensic Science Laboratory, Kolkata, West Bengal, India
2 Office of Director, Central Forensic Science Laboratory, Chandigarh, India
|Date of Submission||22-Jul-2021|
|Date of Decision||30-May-2022|
|Date of Acceptance||31-May-2022|
|Date of Web Publication||02-Sep-2022|
Division of Biology, Central Forensic Science Laboratory, 30, Gorachand Road, Kolkata - 700 014, West Bengal
Source of Support: None, Conflict of Interest: None
Among different endangered animal species, snakes are the most neglected creature looked at with apathy and therefore, are ruthlessly killed, illegally trafficked, and poached for their venom, lucrative skin, meat, and bones for manufacturing of medicines, accessories, and food items. Establishing the identity of the endangered snake species is important for punishing the offenders under Wildlife Protection Act (WPA) (1972) but morphological characters fail to establish identity as they are often altered. The technique of identification of snake species at molecular level holds very effective conclusion in punishing offender. Here, we have constructed and demonstrated a novel multiplexing polymerase chain reaction technique, using 16S rRNA and C-mos gene for identification of four Indian snake species, namely Ptyas mucosa, Daboia russellii, Naja naja, and Xenochrophis piscator. They are listed in Appendix-II and III of convention on international trade in endangered species of wild fauna and flora and Schedule II; Part II of Indian WPA, 1972. Therefore, it may be considered a functional tool for establishing species-specific identity of four Indian snake species and promising to be useful for their conservation.
Keywords: 16S rRNA, C-mos, forensic identification, Indian snakes, multiplex polymerase chain reaction amplification, Wildlife Protection Act (1972)
|How to cite this article:|
Mitra I, Roy S, Haque I. Forensic identification of four Indian snake species using single multiplex polymerase chain reaction. J Forensic Sci Med 2022;8:81-7
|How to cite this URL:|
Mitra I, Roy S, Haque I. Forensic identification of four Indian snake species using single multiplex polymerase chain reaction. J Forensic Sci Med [serial online] 2022 [cited 2022 Sep 27];8:81-7. Available from: https://www.jfsmonline.com/text.asp?2022/8/3/81/355565
| Introduction|| |
Snakes (Serpents) are long, elongated, legless, carnivorous reptiles having attractive body color. They exhibit phenotypically diverse patterns with over 3000 species worldwide., They form the most divergent order squamata and their speciation occurred over a relatively short period. In India, snakes are not treated as important creatures but their body parts are illegally trafficked from India to all South-East Asian countries because of the lucrative value of skin, bones, and meat resulting in the depletion of their numbers in Indian bio-reserves. This issue requires serious attention in the context of their conservation and protection. “Wildlife Protection Act (WPA) (1972)” has the sufficient provisions for preserving these precious creatures. In addition, the “convention on international trade in endangered species of wild fauna and flora” (CITES) also regulates the trade of snakes worldwide. Thus, it can be said that there are enough provisions in the law to curb the illicit trade, killing, and poaching of these animals. However, punishment of animal poachers and traffickers depends on identification of species of animal. In the absence of scientific proof, it is difficult to implement the “WPA (1972)” law. The conventional identification technologies based on morphological features are well established, but all are time-consuming. Furthermore, snake skins are treated with chemicals and physically altered, which render morphological and serological examination of species difficult leading to misidentification resulting in lesser punishment and continuous depletion of endangered species. Therefore, in order to impose appropriate wildlife laws, it is time to invent reliable and fast molecular identification techniques to identify the exact species of traded animals. Hence, it is essential to develop modern molecular identification techniques to authenticate seized animals up to species level.
Two venomous and two nonvenomous snakes species namely, “Daboia russellii,” “Naja naja,” “Ptyas mucosa,” and “Xenochrophis piscator” listed in “Appendix-II and III” of “CITES” are widely distributed in the wild and are frequently poached and trafficked for their venom, meat and skin from India. Some characteristics of these 4 snake species are described below.
- Daboia russelii: Also known as Russell's viper or Chandroborha, this is one of the large venomous snakes found in India. They are the most bite-causing and death-causing snake found in the Indian subcontinent., Being a venom-producing species, its population in Indian wildlife is decreasing day by day. They are famous for their large spitting range. Hugely trafficked from India to core South-East Asian countries for medicinal purposes
- Naja naja: Also known as Indian cobra or Spectacled cobra or Gokhro, this is a venomous snake species which is a member of the big four snakes of India. They are famous for their venom and get poached and illegally trafficked for it
- Ptyas mucosa: Also known as the Indian rat snake or Dhaman, this is a nonvenomous snake which is mistakenly killed for its similarity with Indian Cobra or Naja naja. Apart from this, it is a large size snake and huge lucrative value is attached to its skin for their illegal trafficking
- Xenochrophis piscator: Commonly known as Asiatic water snake and a nonvenomous species with attractive textured skin and are killed for their meat and skin.,
Various approaches have been made till date for identification of the seized snake exhibit up to species level for proper application of “WPA (1972).” The development of DNA-based identification markers has revolutionized wildlife forensic examination thereby conservation of species. They are powerful tools to examine genetic characteristics of an individual, or an entire population, or a species., These markers could also determine genetic diversity, breeding line, phylogenetic tree, etc., showing their application in the management of the biodiversity conservations and genetic improvement programs. Molecular markers in forensic examination are used for the detection of variations in nucleotide sequences using polymerase chain reaction (PCR) technique. There are many reports that state use of these molecular markers for the identification of plants, reptiles, birds or mammals, in terms of conservation and wildlife forensic identification purposes.,, These techniques are reasonably sensitive, specific and cost-effective, compared to other DNA based assays. However, fast and cost effective methods are always preferred for examination. Therefore, in this study, we have generated two sets of species-specific multiplex PCR markers from “16S ribosomal RiboNucleic Acid” or “16S rRNA” and “Cellular Murine Oocyte Sarcoma” or “C-mos” genes for identification of four commonly found and poached snakes in India.
The 16S rRNA is a mitochondrial DNA gene which encodes for a large subunit of mitochondria or mt LSU. Approximately 1500 base pairs in length. And can be found in both prokaryotic and eukaryotic cells. They are conservative in nature, shows a lack of introns and relative abundance in cells. Contain hypervariable regions that can provide species-specific sequences useful for identification of species. Hence, it has been extensively used for phylogenetic study of a species., It can be a valuable tool for species identification of extremely degraded exhibit samples. On the other hand, the nuclear C-mos gene is a single copy without introns and is just over 1000 base pairs. It has been found in the genomes of amphibians, birds, mammals, and reptiles. It acts as proto-oncogene that encodes for serine/threonine kinase and regulates oocyte maturation at meiotic metaphase II. There are no repetitive elements in the sequences and less number of insertions or deletions would complicate sequence alignment among vertebrates. These characteristics make this gene very compliant to PCR amplification from genomic DNA and direct sequencing of PCR products. The amplified products are then identified using simple 2% Agarose Gel electrophoresis techniques. The species-specific PCR assay is considered vital for the identification of four-snake species in this study.
| Materials and Methods|| |
Ethical approval statement of the study
Experiments were carried out in accordance with the ethical protocol of RandD Board of Central Forensic Science Laboratory, Kolkata, West Bengal, Government of India, and in accordance with the permission from the Ministry of Environment and Forests (Wildlife Divisions), Government of India, New Delhi, India, with Approval Letter No.-Ref: F. No. 1-28/20015 WL-I dated: May 21, 2015 and Principal Conservator and Chief Wildlife Warden, West Bengal, India, with Approval Letter No.-Memo No. 3843/WL/4R-6/2015; dated July 13, 2017.
The biological samples of “Indian Rat snake” or “Ptyas mucosa” (15n), “Russell 's viper” or “Daboia russellii” (15n) and “Checkered keelback” (15n) or “Xenochrophis piscator” (15n) were collected from Alipore Zoological Garden, Kolkata, West Bengal, India; Wildlife Rescue Centre and Transit Facility, Salt Lake, Kolkata, West Bengal, India and Snake transit house, Jabalpur, Madhya Pradesh, India. The other species (Crocodile, Lizards, and Fish) for specificity testing were obtained from the repository of Central Forensic Science Laboratory, Kolkata, India, which were collected from Madras Crocodile Bank Trust, Mamallapuram, Chennai, India, and Alipore Zoological Garden, Kolkata, West Bengal, India.
Extraction of genomic DNA and quality check
From shed skin samples DNA was extracted using “Fetzner (1999) protocol.” From tissue samples whole DNA was extracted using “Qia tissue DNA extraction kit” (Qiagen, Valencia) as per the manufacturer's procedure.
DNA quality and quantity were checked using 1% Agarose gel electrophoresis [Figure 1]. For this we used one blank with only water and one control sample of 100 ng/μl along with isolated DNA in the gel block and run the electrophoresis. The brighter the fluorescence, the higher the concentration, i.e., >100 ng/μl, and vice-versa.
|Figure 1: 1% Agarose gel electrophoresis.; A: Control DNA of 100ng/μl; B: Ptyas mucosa; C: Daboia russellii; D: Naja naja; E: Xenochropis piscator; F: Water|
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Polymerase chain reaction amplification and sequencing
At first, PCR amplification of the targeted 16s rRNA and C-mos genes were performed using established forward and reverse primers., Standard PCR procedures were performed using 10 × PCR Buffer, 5 mM MgCl2, 200 μM dNTPs, 1U/μl Taq polymerase (Applied Biosystem), 1.25 μM forward and reverse primers of both the gene markers and 20 ng/μl extracted genomic DNA. The final reaction volume was set as 20 μl. PCR cycles were set as, “initial denaturation” at 94°C for 4 min, followed by 38 cycles of “denaturation” at 94°C for 30 s, “primer annealing” at 56°C for 16S rRNA primers, and 60.5°C for C-mos primers for 1 min 30s, “primer extension” at 72°C for 7 min, then “post cycling extension” at 72°C for 10 min followed by hold at 4°C. The amplified PCR products were detected by 2% Agarose gel electrophoresis methods, using ethidium bromide stain (0.5 μg/ml) [Figure 2] and [Figure 3]. The 16S rRNA gene showed amplifications around 450 bp and C-mos gene showed amplifications around 700 bp. All the amplified products were then cycle sequenced using BigDye Terminator Cycle sequencing kit v 3.1 (Applied Bio-systems, Foster City, CA). DNA sequencing of PCR products was performed on ABI Prism 3100 Genetic Analyzer.
|Figure 2: 2% Agarose gel electrophoresis using 16S rRNA universal primer which shows fluorescence at 450 bp. A: Ptyas mucosa; B: Daboia russellii; C: Naja naja; D: 1kb Ladder; E: Water|
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|Figure 3: 2% Agarose gel electrophoresis using C-mos universal primer primer which shows fluorescence at 700 bp. A: 1kb Ladder; B: Water; C: Ptyas mucosa; D: Daboia russellii; E: X. piscarator|
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Species-specific multiplex polymerase chain reaction primer design
We used two genes to design two sets of species-specific multiplex-PCR primers. All the sequences for both the genes of studied snake species were obtained from available public database (National Centre for Biotechnology Information) and the accession numbers are given in [Table 1] and [Table 2]. To identify species-specific positions, we aligned those sequences of snake species along with the partially sequenced gene in our laboratory and aligned them using MEGA 7 software. To design species-specific reverse primers, interspecific nucleotide sequence differences were observed. These reverse primers were paired with the newly designed forward primer for both the genes (16S and C-mos) and amplified three different snake species through each PCR set. [Table 3] and [Table 4] show the designed common primer and species-specific reverse primers. The multiplex-PCR assays were developed to differentiate all four-snake species with two sequential panels. Strategies for designing both the multiplexing primers are shown in [Figure 4] and [Figure 5].
|Table 1: List of snake species along with their National Centre for Biotechnology Information GenBank accession numbers that we have used while designing 16S rRNA gene multiplex primers|
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|Table 2: List of snake species along with their National Centre for Biotechnology Information GenBank accession numbers that we have used while designing C-mos gene multiplex primers|
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|Table 3: Newly constructed 16S rRNA gene primers (Set 1) along with their amplicon size|
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|Table 4: Newly constructed C.mos gene primers (Set 2) along with their amplicon size|
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Multiplex polymerase chain reaction amplification
Two sets of single-tube multiplex PCR amplifications were carried out to detect different snake species groups simultaneously. The Multiplex PCR reactions were carried out using 10x PCR buffer, 5 mM MgCl2, 0.2 mM dNTPs, 1.50 μm forward, and 1.50 μm species-specific reverse primers, 1.0U/μl of Taq polymerase (Applied Biosystem), and 20 ng of extracted DNA. PCR cycles were set as follows; “initial denaturation” at 94°C for 4 min, followed by 38 cycles of “denaturation” at 94°C for 30 s, species-specific “primer annealing” temperature (at 56°C for 16S rRNA gene and 60°C for C-mos gene), for 1 min 30 s, “primer extension” at 72°C for 1 min 15 s, then post cycling or “final extension” at 72°C for 7 min followed by final hold at 4°C. The final amplified PCR products were detected by 2.5% Agarose gel electrophoresis methods, using ethidium bromide stain (0.5 μg/ml) [Figure 6]a and [Figure 6]b.
|Figure 6: (a) 2.5% agarose gel electrophoresis results using 16MCF, 16MPMR, 16MNNR and 16MDRR primers designed in this study. A: Water; B: 1Kb Ladder; C: Single tube multiplex; D: Naja naja (180 bp); E: Ptyas mucosa (50 bp); F: Daboia russellii (200 bp). (b) 2.5% agarose gel electrophoresis results using CMCF, CMPMR, CMXPR and CMDRR primers designed in this study. A: 1Kb Ladder; B: Single tube multiplex; C: Xenochrophis piscator (120 bp); D: Daboia russellii (150 bp); E: Ptyas.mucosa (250 bp); F: Water|
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| Results|| |
Multiplex PCR is a variant of PCR process where two or more loci are simultaneously amplified in the same run. Here, a common forward primer and three (3) species-specific reverse primers have been constructed for both the genes, separately, to amplify three different size species-specific amplicons. Both the multiplex-PCR panel shows the rapid identification of individual species assigned to each of them.
First, two single-tube multiplex PCR reactions [panel A for [Figure 6]a and panel B for [Figure 6]b] were performed using species-specific three reverse primers 16MPMR (16S rRNA Multiplex Ptyas mucosa Reverse), 16MNNR (16S rRNA Multiplex Naja naja Reverse), 16MDRR (16S rRNA Multiplex Daboia russellii Reverse) and CMPMR (C-mos Multiplex Ptyas mucosa Reverse ), CMDRR (C-mos Multiplex Daboia russellii Reverse), CMXPR (C-mos Multiplex Xenochrophis piscator Reverse) in combination with common forward primer 16MCF or CMCF (16S rRNA common forward or C-mos common forward), allowing the following amplifications, visible under 2.5% Agarose gel electrophoresis run for 30 min:
For multiplex PCR using 16S rRNA marker [Figure 6]a:
- A single band of 180 bp for Naja naja (D)
- A single band of 50 bp for Ptyas mucosa (E)
- A single band of 200 bp for Daboia russellii (F).
For multiplex PCR using C-mos marker [Figure 6]b:
- A single band of 120 bp for Xenochrophis piscator (C)
- A single band of 150 bp for Daboia russellii (D) and
- A single band of 250 bp for Ptyas mucosa (E).
Second, parallel specificity runs were also performed through individual PCR amplification [Panel D, E, and F for [Figure 6]a and panel C, D, and E for [Figure 6]b] using species-specific reverse primers and forward primers for each sample. The results show the DNA bands occurred at expected length from all the studied species [Figure 6]a and [Figure 6]b.
| Discussions|| |
Forensic DNA testing is a key investigative tool for combating wildlife crimes., Conservation biologists also focus on the progress of novel DNA-based technologies for wildlife protection strategies., Since the inception of the Multiplex PCR technique, it has been successfully applied in many arena of Forensic DNA testing. Although many PCR- restriction fragment length polymorphism techniques and basic local alignment search tool (BLAST) search of sequenced products after universal primer processing methods are available for snake species identification, which is more time-consuming and expensive process. The Multiplex PCR amplification involves simultaneous running of multiple PCR primers or target markers in a single-tube PCR reaction. It was designed for speedy identification of species without former sequencing., Species-specific multiplex PCR markers were used to analyze the four-snake species samples that we studied.,, The multiplex-PCR assays were developed to differentiate all four-snake species with two sequential panels. The designed primers were subject to BLAST search using BLASTx and BLASTn program. The results showed expected amplification at the desired place. These sets of primers are the first DNA markers to exactly identify the four-snake species examined. Our multiplex PCR assay clearly identified four-snake species (Ptyas mucosa, Daboia russellii, Naja naja, and Xenochrophis piscator) in two simultaneous runs followed by 2.5% Agarose gel electrophoresis check. The specificity of each primer for each species was tested in separate reactions. The validation studies were carried out using targeted and nontargeted species DNA with sufficient numbers of individuals from a single species. Therefore, these markers can be effectively used for rapid and accurate identification of species in wildlife forensic casework, conservation studies, and medicinal research.
| Conclusions|| |
Here, we described two Multiplex-PCR reaction kits based on two separate multiplex PCR marker sets which can be used for the rapid identification of all the four Indian snake species. Species identification of the four common Indian snake species has been established by performing PCR programming followed by 2.5% agarose gel electrophoresis which is definitely economic, fast, and accurate. This method of species identification using Multiplex–PCR techniques described in this paper is cost-effective and less time-consuming compared to other techniques based on DNA sequencing. The Multiplex-PCR assays developed in this study could provide a powerful discriminatory tool for forensic identification purposes. Therefore, it is expected that the newly developed Multiplex-PCR assay technique can be used in Wildlife Forensic cases involving indigenous snake samples and help the forensic scientists for providing proper identification of the exhibits from four Indian snake species.
The authors would sincerely like to acknowledge the Directorate of Forensic Science Services, Government of India, India, for giving the opportunity to work on Wildlife Forensics at Central Forensic Science Laboratory, Kolkata, West Bengal, India, and Chief Principal Conservator cum Chief Wildlife Warden, Kolkata, West Bengal, India, to allow us for snake sample collections from Zoological Garden, Alipore, Kolkata, West Bengal, and Wildlife Rescue Centre, Kolkata, West Bengal. We also would like to thank the Anthropological Survey of India, Kolkata, West Bengal, for providing a facility to standardize the PCR reactions using Gradient PCR machine.
All the authors have contributed to the collection of literature and reflected their work experience on wildlife case handling in the article. I H and I M conceptualized the outline of the work. Review of literature, sample collection, experiments, analysis of data, and drafting of the manuscript done by I M. Simultaneously S R, and I H contributed to data analysis and manuscript editing along with I M.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Secor SM, Diamond JM. A vertebrate model of extreme physiological regulation. Nature (London) 1998;395:659-62.
Cox MJ, Hoover MF, Lawan C, Kumthorn T. The snakes of Thailand. Bangkok: Chulalongkorn University Museum of National History; 2012.
Wijnstekers W. The evolution of CITES: A reference to the Convention on International Trade in Endangered Species of Wild Fauna and Flora. 18th
ed. Châtelaine-Geneva, Switzerland: CITES Secretariat 2001.
Avise JC. Molecular Markers, Natural History, and Evolution. New York: Chapman & Hall; 1994. p. 511.
Mallow D, Ludwig D, Nilson G. True Vipers: Natural History and Toxinology of Old World Vipers. Malabar, Florida: Krieger Publishing Company; 2003. p. 359.
Herpetologists' League. Snake Species of the World: A Taxonomic and Geographic Reference. Vol. 1. Washington, District of Columbia: Herpetologists' League; 1999. p. 511.
Vogel G. Terralog: Venomous Snakes of Asia. 1st
ed., Vol. 14. Frankfurt Am Main: Hollywood Import & Export; 2006. p. 148.
Linda KP, Paul M. Developments in molecular genetics techniques in fishery. In: Carvalho GR, Pitcher TJ, editors. Molecular Genetics in Fisheries. London: Chapman and Hall; 1995. p. 1-28.
Hillis DM, Mable BK, Moritz C. Applications of molecular systematic: The state of the field and a look to the future. In: Hillis DM, Mable BK, Moritz C, editors. Molecular Systematics. 2nd
Ed. Sunderland, MA: Sinauer; 1996. p. 515-43.
Meyer R, Candrian U, Lüthy J. Detection of pork in heated meat products by the polymerase chain reaction. J AOAC Int 1994;77:617-22.
Cocolin L, D'Agaro E, Manzano M, Lanari D, Comi G. Rapid PCR-RFLP method for the identification of marine fish fillets (Seabass, Seabream, Umbrine, and Dentex). J Food Sci 2000;65:1315-7.
Arslan A, Ilhak OI, Calicioglu M. Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat Sci 2006;72:326-30.
Magnussen JE, Pikitch EK, Clarke SC, Nicholson C, Hoelzel AR, Shivji M. Genetic tracking of basking shark products in international trade. Animal Conserv 2007;10:199-207.
Mane BG, Tanwar VK, Girish PS, Dixit VP. Identification of species origin of meat by RAPD-PCR technique. J Vet Public Health 2006;4:87-90.
Flook PK, Rowell CH. The effectiveness of mitochondrial rRNA gene sequences for the reconstruction of the phylogeny of an insect order (Orthoptera). Mol Phylogenet Evol 1997;8:177-92.
Naga JK, Meganathan PR, Dubey B, Haque I. Mitochondrial 16S rRNA gene for Forensic identification of crocodile species. J Forensic Legal Med 2013;20:334-8.
Gebauer F, Richter JD. Synthesis and function of Mos: The control switch of vertebrate oocyte meiosis. Bioessays 1997;19:23-8.
Palumbi SR, Cipriano F. Species identification using genetic tools: The value of nuclear and mitochondrial gene sequences in whale conservation. J Hered 1998;89:459-64.
Fetzner JW Jr. Extracted high-quality DNA from shed reptile skins: A simplified method. BioTechniques 1999;26:1052-54.
Palumbi SR, Martin A, Romano S, McMillan WO, Stice L, Grabowski G. The Simple Fool's Guide to PCR, Version 2. Honolulu: University of Hawaii Zoology Department; 1991.
Saint KM, Austin CC, Donnellan SC, Hutchinson MN. C-mos, a nuclear marker useful for squamate phylogenetic analysis. Mol Phylogenet Evol 1998;10:259-63.
Coordinators NR. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 2018;46:D8-13.
Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4.
Iyengar A. Forensic DNA analysis for animal protection and biodiversity conservation: A review. J Nat Conserv 2014;22:195-205.
Ibrahim AA, Haseeb AK, Ali HB. DNA marker technology for wildlife conservation. Saudi J Biol Sci 2011;18:219-25.
Kumar A, Walker S, Molur S. Prioritization of Endangered Species. Setting Biodiversity Conservation Priorities for India. New-Delhi: WWF-India; 2000. p. 341-425.
Ogden R, Dawnay N, McEwing R. Wildlife DNA forensics-bridging the gap between conservation genetics and law enforcement. Endangered Species Res 2009;9:179-95.
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: Critical parameters and step-by-step protocol. Biotechniques 1997;23:504-11.
Unajak S, Meesawat P, Anyamaneeratch K, Anuwareepong D. Identification of species (meat and blood samples) using nested-PCR analysis of mitochondrial DNA. Afr J Biotechnol 2011;10:5670-6.
Dubey B, Meganathan PR, Haque I. Multiplex PCR assay for rapid identification of three endangered snake species of India. Conserv Genet 2009;10:1861-4.
Meganathan PR, Dubey B, Jogayya KN, Haque I. Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles. J Forensic Sci 2011;56:1241-4.
Datukishvili N, Kutateladze T, Gabriadze I, Bitskinashvili K, Vishnepolsky B. New multiplex PCR methods for rapid screening of genetically modified organisms in foods. Front Microbiol 2015;6:757.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]