猪腹泻病毒一步法多重TaqMan荧光定量RT-PCR检测法的建立及应用

doi:10.3864/j.issn.0578-1752.2023.01.014

Abstract

Abstract:

【Objective】 The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. 【Method】The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value.The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. 【Result】 Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1×10² copies/μL. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. 【Conclusion】In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.

Key words: one-step multiple TaqMan real-time RT-PCR, swine diarrhea related virus, differential diagnosis, cut off value

WANG YiDan,YANG FaLong,CHEN DiShi,XIANG Hua,REN YuPeng. One-Step Multiple TaqMan Real-time RT-PCR for Simultaneous Detection of Swine Diarrhea Viruses[J].Scientia Agricultura Sinica, 2023, 56(1): 179-192.

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References 28

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病原 Pathogen	样本类型 Sample type	来源 Source ¹⁾	总数 Total
PEDV SCXM	核酸 Nucleic acid	a	1
PEDV SWUN-D1	核酸 Nucleic acid	a	1
PEDV SWUN-D2	核酸 Nucleic acid	a	1
GARV G3	核酸 Nucleic acid	a	1
GARV G5	核酸 Nucleic acid	a	1
GARV G9	核酸 Nucleic acid	a	1
PDCoV	核酸 Nucleic acid	a	1
PTV 3	核酸 Nucleic acid	a	1
PTV 6	核酸 Nucleic acid	a	1
PEDV CV777	疫苗株 Vaccine strains	b	1
PEDV AJ1102	疫苗株 Vaccine strains	c	1
TGEV	核酸 Nucleic acid	a	1
CSFV	核酸 Nucleic acid	a	1
PRV	核酸 Nucleic acid	d	1
PRRSV SCMY2018	分离株 Isolated strains	d	1
S.choleraesuis SWUN-A6	分离株 Isolated strains	a	1
P.multocida SWUN-MY	分离株 Isolated strains	a	1
E.coli SC-09	分离株 Isolated strains	a	1
S.suis SWUN-10	分离株 Isolated strains	a	1
S.aureus SWUN-C2	分离株 Isolated strains	a	1

病原 Pathogen	基因 Gene	引物序列 Primer sequence (5′-3′)	片段长度 Size (bp)
PEDV	ORF3	F: TGTTGTAGGGGTCCTAGACTTC	887
PEDV	ORF3	R: CTATACAAACGCCCTATAGGTG	887
GARV	NSP5	F: ATGTCTCTCAGCATTGACGTA	630
GARV	NSP5	R: GAGTGGGGAGCTCCCTAGTGT	630
PDCoV	M	F: ATGTCTGACGCAGAAGAGTG	974
PDCoV	M	R: CTACTGGTGCGGCCATGATAG	974
PTV	5'UTR	F: ACTCGTTACGCAAGTTTTGG	348
PTV	5'UTR	R: CAAAGTACAGACGGTCAGCC	348
SADS-CoV	N	F: ATGGCCACTGTTAATTGGG	1113
SADS-CoV	N	R: ATCCACCATCTCAACCTCC	1113

病原 Pathogen	基因 Gene	引物及探针序列 Primer and probe (5′-3′)	片段长度 Size (bp)
PEDV	ORF3	F: TTTAAAGCGTCTTCTTTGMG	177
		R: ATTTTTATAGCGCCAKGAG
		P: VIC- CTGTCRTYKTTCTTTATCGCCCACTT-BHQ1
GARV	NSP5	F: GGTAGGAGTGAACAGTACA	140
		R: GYGTYGATGAATCCATAG
		P: ROX-TTAAGACAAATGCAGAYGCTGGCG-BHQ2
PDCoV	M	F: CACATGGCTCCAATTCTC	198
		R: TGGATCGCTGTTTGATTC
		P: FAM-TCGTTAAGCATGGCAAGCTCAAGCT-BHQ1
PTV	5'UTR	F: AAYTGTCACCRGGTAYTG	151
		R: TGYATTCCSATACAGGAA
		P: CY5-AGAAGAGCAAGTACTCCTGACTGGG-BHQ2
SADS-CoV	N	F: AATGAAGGTCCCCAAGAC	166
		R: AGGATTTTGGGAAACTGG
		P: Cy5.5-ATGCTGATGCCCCAGTGTTCACT- BHQ3

浓度 Concentration (copies/μL)	CT值 Cycle threshold value
浓度 Concentration (copies/μL)	PEDV	GARV	PDCoV	PTV	SADS-CoV
1×10⁸	15.24	15.13	15.43	12.83	14.54
1×10⁷	20.36	18.01	17.73	15.73	17.55
1×10⁶	22.8	21.49	22.23	18.75	21.01
1×10⁵	26.52	24.54	25.1	22.31	24.42
1×10⁴	29.52	27.74	28.44	25.87	27.68
1×10³	32.55	31.91	31.01	28.88	31.31
1×10²	34.84	34.19	34.91	32.82	35.00
1×10¹	None	None	None	None	37.42

病原 Pathogen	稀释倍数 Dilution (copies/μL)	组内 Intra-assay		组间 Inter-assay
病原 Pathogen	稀释倍数 Dilution (copies/μL)	Ct值Ct value (mean ± SD)	变异系数 CV (%)	Ct值Ct value (mean ± SD)	变异系数 CV (%)
GARV	1×10⁸	15.04±0.18	1.22%	15.27±0.09	1.99%
	1×10⁵	24.58±0.05	0.22%	24.29±0.047	0.89%
	1×10²	32.59±0.75	2.31%	33.03±0.099	0.95%
PEDV	1×10⁸	15.33±0.41	2.64%	15.76±0.11	2.07%
	1×10⁵	25.44±0.27	1.06%	25.53±0.19	1.69%
	1×10²	33.93±0.75	2.20%	34.14±0.03	0.47%
PDCoV	1×10⁸	14.38±0.35	2.46%	14.65±0.29	3.68%
	1×10⁵	22.43±0.46	2.04%	23.46±0.88	4.00%
	1×10²	35.03±0.68	1.95%	34.82±0.13	1.03%
PTV	1×10⁸	12.97±0.22	1.71%	13.19±0.23	3.66%
	1×10⁵	23.3±0.41	1.76%	23.05±0.04	0.91%
	1×10²	30.01±0.13	0.42%	31.51±1.43	3.80%
SADS-CoV	1×10⁸	14.98±0.46	3.08%	14.99±0.07	1.78%
	1×10⁵	24.82±0.29	1.16%	25.06±0.037	0.77%
	1×10²	35.34±0.45	1.28%	35.68±0.13	1.00%

One-Step Multiple TaqMan Real-time RT-PCR for Simultaneous Detection of Swine Diarrhea Viruses

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			其他同类方法 The reference detection method			符合率 Coincidence rate (%)
			Positive	Negative	Total	符合率 Coincidence rate (%)
一步法多重 TaqMan 荧光定量RT-PCR One-step multiple TaqMan real-time RT-PCR for simultaneous detection of five swine diarrhea viruses	PEDV	Positive	26	0	26	97.9% (k=0.9)
		Negative	5	211	216
		Totals	31	211	242
	GARV	Positive	30	3	33	98.8% (k =0.95)
		Negative	0	209	209
		Totals	30	212	242
	PDCoV	Positive	44	0	44	100% (k =1)
		Negative	0	198	198
		Totals	44	198	242
	PTV	Positive	31	4	35	98.3% (k =0.93)
		Negative	0	207	207
		Totals	31	211	242
	SADS-CoV	Positive	0	0	0	100% (k =1)
		Negative	0	0	242
		Totals	0	242	242

样本来源 Source	五种猪腹泻病毒检出率 The detection rates of five kinds of porcine diarrhea virus
样本来源 Source	PEDV	GARV	PDCoV	PTV	SADS-CoV
乐至 Lezhi	30% (3/10)	0% (0/10)	40% (4/10)	10% (1/10)	0% (0/10)
雅安 Yaan	19% (4/21)	14.3% (3/21)	0% (0/21)	0% (0/21)	0% (0/21)
恩阳 Enyang	0% (0/10)	30% (3/10)	0% (0/10)	20% (2/10)	0% (0/10)
高坪 Gaoping	0% (0/10)	40% (4/10)	30% (3/10)	30% (3/10)	0% (0/10)
绵阳 Mianyang	9.9% (19/191)	12% (23/191)	19.4% (37/191)	15.2% (29/191)	0% (0/191)
总计 Total	10.7% (26/242)	13.6% (33/242)	18.2% (44/242)	14.5% (35/242)	0% (0/242)