猪圆环病毒2型和弓形虫双重PCR检测方法的建立及应用
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S855.3

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贵州省科技支撑计划项目(2020-1Y032)


Establishment and Application of a Duplex PCR for Porcine Circovirus Type 2 and Toxoplasma gondii
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    摘要:

    为建立一种同时检测猪圆环病毒2型(PCV2)和弓形虫(Toxoplasma gondii)的双重PCR方法,根据GenBank上已登录的PCV2和弓形虫基因的保守序列,设计了2对特异性引物,并对反应体系及条件进行了优化。首先对单一PCR检测条件进行摸索,确定其最佳检测条件和灵敏性;进而建立两种病原的双重PCR检测方法,通过对退火温度、灵敏性和特异性进行测试,最终确定该方法的反应体系及条件。结果显示:双重PCR方法对PCV2和弓形虫的最低检出限分别为78.16和29.19 μg/mL;对伪狂犬病毒(PRV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)等均无扩增,特异性良好。利用该方法对来自川渝地区部分猪场的63份临床样本进行检测,并与单一PCR检测结果进行比对,两种方法的符合率为100%。以上结果说明,本试验建立的双重PCR方法灵敏度高、特异性强、结果可靠、方便经济,可用于PCV2和弓形虫的实验室快速检测。

    Abstract:

    In order to develop a duplex PCR assay for porcine circovirus type 2(PCV2)and Toxoplasma gondii,two pairs of specific primers were designed based on the conserved sequences of the genes of PCV2 and Toxoplasma gondii registered in GenBank,then their reaction systems and conditions were optimized. Firstly,the single PCR detection conditions were tested to determine the optimal detection conditions and sensitivity;then a duplex PCR assay was established to detect the two pathogens,and its reaction system and conditions were finally determined by testing the annealing temperature,sensitivity and specificity. The results showed that the lowest detection limits of the established method for PCV2 and Toxoplasma gondii were 78.16 μg/mL and 29.19 μg/mL,respectively;no amplification was observed for pseudorabies virus(PRV),porcine parvovirus(PPV),porcine reproductive and respiratory syndrome virus(PRRSV)and classical swine fever(CSFV),indicating a good specificity. 63 clinical samples collected from Sichuan-Chongqing region were detected by the established method,and the results were compared with those obtained by singe PCR. It was found that the coincidence rate of the two methods was 100%. In conclusion,the duplex PCR established in the experiment was with high sensitivity,strong specificity,reliable results,convenience and low cost,which could be used for rapid detection of the above two pathogens by laboratories.

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朱桓奕,罗亮,张璇,李照伟,浦同灿,王慧,曾红,杨晓伟,刘霞,赵光伟.猪圆环病毒2型和弓形虫双重PCR检测方法的建立及应用[J].《中国动物检疫》编辑部,2022,39(11):111-114.

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  • 在线发布日期: 2022-11-03
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