Development of novel heminested PCR assays based on mitochondrial 16s rRNA gene for identification of seven pecora species

Table 3 Results of validation study of species-specific heminested PCR assays

Validation Parameters		No. of samples analysed	No. of specific primers analysed	No. of samples successfully typed
DNA concentration	5pg	20	7	20
	50 pg	20	7	20
	500 pg	20	7	20
	5 ng	20	7	20
Mixture analysis ^$	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^a	1 (Blackbuck)
	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^b	1 (Nilgai)
	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^c	1 (Goral)
	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^d	1 (Hog deer)
	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^e	1 (Chital)
	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^f	1 (Thamin)
	Blackbuck + Nilgai + Goral + Hog deer + Chital + Thamin + Sambar	1 (mixture)	1^g	1 (Sambar)
Chemical reagents	Soap	20	7	20
	0.1 N NaOH	20	7	20
	5% Acetic acid	20	7	20
	1 M NaCL	20	7	20
	Gasoline	20	7	20
Exposure to Heat	37°C, 1 week	7*	7	7
	37°C, 2 week	7*	7	7
	37°C, 3 week	7*	7	7
UV Irradiation (302 nm, 15 cm distance)	1 h	20	7	20
	8 h	20	7	20
	24 h	20	7	18

* One sample of each species were included for Exposure to Heat validation
^$ Each mixture comprised 50 pg of DNA of target species and 500 pg of DNA of each non-target species a – Blackbuck specific primer, b – Nilgai specific primer, c- Goral specific primer, d – Hog deer specific primer, e – Chital specific primer, f – Thamin specific primer, g – Sambar specific primer.

ISSN: 2730-6844