Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection

Table 3 Test validation performed on viral RNA extractions with predetermined but unknown status

Sample status^a	Identifier	Test
		Melting temperature	SYBR Green real time RT-PCR	Conventional RT-PCR	Sequencing	Quantification (genomic copies/μl of RNA extraction)
PoSaV +	PC44^b	80.5	-	-	ND
	PC45	86.0	+	+	+	1.41E+05
	PC46	86.0	+	+	+	3.25E+04
	PC47	85.0	+	+	+	2.91E+04
	PC48	87.5	+	+	+	2.76E+05
	PC49	86.0	+	+	+	1.04E+06
PoNoV +	PC54	84.5	-	+/−^c	-
	PC55	80.0	-	+/−^c	-
	PC58	80.0	-	+/−^c	-
	PC59 ^b	81.5	-	-	ND
	PC60	80.5	-	+/−^c	-
	PC61	80.5	-	+/−^c	-
	PC62	84.5	-	+/−^c	-
Negative	PC52	81.5	-	-	ND
	PC53	79.5	-	-	ND
	PC56	80.0	-	-	ND
	PC57	82.0	-	-	ND
	PC63	85.0	+ ^d	+	+

^a: Sample status was determined in another laboratory. Positive porcine sapovirus (PoSaV) samples were determined by conventional RT-PCR; positive porcine norovirus (PoNoV) samples were determined by real time RT-PCR.
^b: Low positive in the reference laboratory.
^c: Doubtful samples, as difference between molecular weights of amplicons obtained with p289/290 primer pair on sapovirus- and norovirus-positive amplicons were very small.
^d: The sequencing reaction performed on that sample gave a sequence genetically related to the QW19-like, SWECII/VA103 and SWECII/VA14 porcine sapovirus strains. QW19 strains are known to share high amino acid identities with human sapovirus strains (Wang et al., 2005) and thus, QW19-related strains could provide slightly lower melting temperatures, such as those registered for human sapovirus.
Ct: Cycle threshold; ND: not done; PoSaV: porcine sapovirus; PoNoV: porcine norovirus.

ISSN: 1746-6148