Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3

doi:10.3969/j.issn.1004-1524.2022.03.05

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (3): 457-463.DOI: 10.3969/j.issn.1004-1524.2022.03.05

• Animal Science • Previous Articles Next Articles

Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3

MOU Hongye¹(), ZHOU Xiaojie¹, YANG Yongchun¹, WANG Xiaodu¹, ZHOU Yingshan¹^,²^,^*(), SONG Houhui¹^,^*

1. Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China
2. Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China

Received:2020-11-06 Online:2022-03-25 Published:2022-03-30
Contact: ZHOU Yingshan,SONG Houhui

Abstract

Abstract:

To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3),specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested.Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies·μL^-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181),the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181).The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181).The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.

Key words: porcine circovirus 2, porcine circovirus 3, TaqMan probe, duplex fluorescence quantitative PCR

CLC Number:

S852.65

MOU Hongye, ZHOU Xiaojie, YANG Yongchun, WANG Xiaodu, ZHOU Yingshan, SONG Houhui. Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 457-463.

Figures/Tables 7

Fig.1 Amplification of the target genes by PCR M, DL2000 DNA Marker;1,PCV3 gene; 2,PCV2 gene.

Fig.2 The standard curve of TaqMan fluorescence quantitative PCR

Fig.3 Specificity detection results of duplex fluorescence quantitative PCR 1,PCV2; 2, PCV3; 3,PRRSV; 4,PRV; 5,PPV; 6,CSFV; 7,PEDV; 8,TGEV; 9,ddH2O.

Fig.4 Sensitivity detection results of duplex fluorescence quantitative PCR 1-8,1.0×108-1.0×101 copies·μL-1 of pMD-PCV2; 9,PCV2 negative control; a-h,1.0×108-1.0×101 copies·μL-1 of pMD-PCV3; i, PCV3 negative control.The same as below.

Fig.5 Sensitivity detection results of ordinary PCR 1-8, 1.0×108-1.0×101copies·μL-1 of pMD-PCV2; 9,PCV2 negative control; a-h,1.0×108-1.0×101copies·μL-1 of pMD-PCV3; i, PCV3 negative control.

Table 1 Reproducibility test of PCV2 and PCV3 standard products

质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	组内重复试验(Ct) Intra-assay Ct value		组间重复试验(Ct) Inter-assay Ct value
质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	平均值 $\bar{x}$ ±SD	变异系数CV/%	平均值 $\bar{x}$ ±SD	变异系数CV/%
PCV2	10⁴	28.24±0.15	0.53	27.90±0.19	0.68
	10³	31.67±0.37	1.17	32.06±0.11	0.34
	10²	34.49±0.29	0.84	34.02±0.44	1.29
PCV3	10⁴	26.53±0.13	0.49	27.27±0.16	0.59
	10³	29.15±0.12	0.41	29.21±0.12	0.41
	10²	32.19±0.09	0.28	32.59±0.14	0.43

Table 1 Reproducibility test of PCV2 and PCV3 standard products

质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	组内重复试验(Ct) Intra-assay Ct value		组间重复试验(Ct) Inter-assay Ct value
质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	平均值 $\bar{x}$ ±SD	变异系数CV/%	平均值 $\bar{x}$ ±SD	变异系数CV/%
PCV2	10⁴	28.24±0.15	0.53	27.90±0.19	0.68
	10³	31.67±0.37	1.17	32.06±0.11	0.34
	10²	34.49±0.29	0.84	34.02±0.44	1.29
PCV3	10⁴	26.53±0.13	0.49	27.27±0.16	0.59
	10³	29.15±0.12	0.41	29.21±0.12	0.41
	10²	32.19±0.09	0.28	32.59±0.14	0.43

Table 2 Test results of clinical samples

检测方法 Method	PCV2		PCV3		共感染 Co- infection	共感染率 Co-infection rate/%
检测方法 Method	阳性样品 Number of positive samples	检出率 Detection rate/%	阳性样品 Number of positive samples	检出率 Detection rate/%	共感染 Co- infection	共感染率 Co-infection rate/%
双重荧光定量PCR Double fluorescence quantitative PCR	92	50.83	68	37.57	22	12.15
普通荧光定量PCR Ordinary fluorescence quantitative PCR	91	50.28	66	36.46	21	11.60
符合率Coincidence rate/%	98.91	97.06	95.45

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Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3

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