猪圆环病毒3型TaqMan荧光定量PCR方法的建立和初步应用

doi:10.11843/j.issn.0366-6964.2018.06.026

Abstract

Abstract:

To detect the Porcine Circovirus type 3 (PCV3) sensitively, rapidly and specifically, four pairs of primers were designed targeting the conserved region of PCV3 ORF3 gene. SYBR-based quantitative PCR results showed that Real-time PCR with all primer pairs can detect viral genome efficaciously. Then a TaqMan probe were designed according to the PCR products intersection sequence, and a real-time quantitative PCR method was established by optimizing the reaction conditions and systems. The method presented a good linear relation with the Pmd18-t-Cap vector(Recombinant plasmid harboring PCV3 ORF2 gene) ranging from 1.29×10²-1.29×10⁹ copies·μL^-1. The assay was highly specific for PCV3, without cross-reaction with other porcine virus and bacteria, such as Porcine circovirus 2(PCV2), Porcine reproductive and respiratory syndrome virus(PRRSV), Porcine rotavirus(PoRV), Haemophilus parasuis(Hps), Actinobacillus pleuropneumoniae(App). And the limit detection of this assay was 1.29×10² copies·μL^-1, more sensitive than the conventional PCR. The CV (Coefficient of Variation) of intra-assay and inter-assay were less than 2%, showed that the assay was reproducible. The real-time qPCR was further used to detect the DNA sample extracted from clinical sample collected from 2016 to 2017, the PCV3 positive ratio was 8.06% (10/124), and the co-infection rate of PCV3/PCV2 was 8.06% (10/124). The real-time qPCR assay provides a high sensitivity and specificity, repeatability method for detection of PCV3.

Key words: porcine circovirus type 3(PCV3), SYBR qPCR, TaqMan qPCR, detection

CLC Number:

S852.659.2

LI Chang, KU Xu-gang, WANG Jun-wei, LI Jing, ZHU Ling, HE Qi-gai. Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1320-1326.

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Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3

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