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

摘要/Abstract

摘要：

【目的】建立一种可同时检测猪流行性腹泻病毒（PEDV）野毒株、猪A群轮状病毒（GARV）、猪德尔塔冠状病毒（PDCoV）、猪阿尔法冠状病毒（SADS-CoV）及猪捷申病毒（PTV）5种猪腹泻病毒的一步法多重TaqMan荧光定量RT-PCR检测法。为猪腹泻病的快速诊断和流行病学调查提供高效灵敏的工具。【方法】对 PEDV 多个基因型毒株ORF3基因比对分析，以PEDV野毒株为模板，对疫苗株ORF3基因稳定缺失区域设计特异性探针，并在两端保守区域设计上下游引物；在靠近GARV G3、G4、G5和G9型 NSP5基因5′端保守碱基区域设计引物及探针，并加入简并碱基。同时，分别选择PDCoV M基因、PTV 5′UTR序列、SADS-CoV N基因等保守基因设计特异性引物及探针，用于多重荧光定量PCR方法的建立。对引物、探针浓度和退火温度进行优化；用RStudio参照代码绘制ROC曲线，确定检测方法的敏感度值、特异度值及曲线下面积AUC，并计算Youden指数，最终确定检测临界值；从阳性核酸中扩增靶基因，并克隆至pEASY-T1载体。通过体外转录，获得5种标准品分别命名为：cRNA-PEDV、cRNA-GARV、cRNA-PDCoV、cRNA-PTV和cRNA-SADS-CoV。对检测方法的敏感性、特异性和重复性等进行评估；并与同类方法对临床样本的检测符合率进行比较。【结果】得到了5种病原检测的最佳引物、探针浓度和最佳退火温度。根据ROC曲线确定PEDV、GARV、PDCoV、PTV和SADS-CoV临界CT值分别为：35.78、34.25、34.98、34.60和35.70；5种病原的检测下限均可达到1×10² copies/μL，标准曲线线性关系良好，扩增效率在96.3%—104%之间；该方法对PEDV CV777疫苗株、PEDV AJ1102疫苗株、猪传染性胃肠炎病毒（TGEV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）、猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪霍乱沙门氏菌（S.choleraesuis）、多杀性巴氏杆菌（P.multocida）、大肠杆菌（E.coli）、猪链球菌（S.suis）和葡萄球菌（S.aureus）等多种菌毒株均不检出，具有良好的特异性；经重复性检验，组内变异系数在0.22%—3.08%之间，组间变异系数在0.89%—4.0%之间；对242份临床样本检测并与同类方法的检测结果进行比较，符合率分别为：PEDV 97.9%、GARV 98.8%、PDCoV 100%、PTV98.3%和SADS-CoV100%，Kappa值均大于0.9。对PEDV野毒株的检测准确性高于同类方法。此外，对临床样本检测结果显示，当前四川省腹泻猪群中尚无SADS-COV检出；但PEDV、GARV、PDCoV和PTV仍持续流行，其总体阳性率分别达到：10.7%（26/242）、13.6%（33/242）、18.2%（44/242）和14.5%（35/242），且各腹泻病原之间存在不同形式和程度的混合感染，使感染猪腹泻病情加剧。故需进一步加强几种猪腹泻病毒在本地区猪群中流行情况调查和遗传变异规律研究，为制定更具针对性的防控措施提供依据。【结论】本研究成功建立了一种同时检测PEDV野毒、PDCoV、SADS-CoV和 GARV、PTV多基因型的一步法多重TaqMan荧光定量RT-PCR，为猪腹泻病的快速鉴别诊断和流行病学调查提供了一种高效灵敏的工具。

关键词: 一步法多重TaqMan荧光定量RT-PCR, 猪腹泻相关病毒, 鉴别诊断, 检测临界值

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

王一丹,杨发龙,陈弟诗,向华,任玉鹏. 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[J]. 中国农业科学, 2023, 56(1): 179-192.

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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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%