8种猪呼吸道和繁殖障碍病病原体GeXP检测方法的建立

doi:10.3864/j.issn.0578-1752.2015.24.014

摘要/Abstract

摘要： 【目的】建立了一种同时鉴别H1、H3亚型猪流感、猪繁殖与呼吸障碍综合征、猪瘟、猪日本乙型脑炎、猪圆环病毒病、猪细小病毒病和猪伪狂犬病8种病毒性呼吸道和繁殖障碍病病原体的GeXP 高通量检测方法。【方法】根据这8种病原体的基因保守序列，设计并合成了9对特异性引物, 在每对特异性引物的5′端均加上一段通用引物，形成特异性嵌合引物。运用GeXP单重PCR方法，以单一病毒cDNA/DNA为模板验证引物的可行性；建立GeXP多重PCR方法，以单一病毒cDNA/DNA模板、阳性cDNA/DNA混合模板验证GeXP多重检测体系的特异性和准确性；将含猪圆环病毒、猪细小病毒和猪伪狂犬病病毒靶基因的克隆质粒及体外转录的RNA（H1、H3亚型猪流感、猪繁殖与呼吸障碍综合征、猪瘟、猪日本乙型脑炎）分别梯度稀释为10³，10²，10¹拷贝/µL，运用GeXP多重PCR方法进行单一病原体灵敏度分析；根据单一病原体的灵敏度分析结果，优化各对特异性嵌合引物的工作浓度，将含有体外转录好的6种RNA模板和3种克隆质粒等量混合，将混合物梯度稀释为10⁴,10³,10²,10¹拷贝/µL，运用GeXP多重PCR方法分析同时检测8种病原体的灵敏度。运用建立好的GeXP多重检测体系对23份临床样品进行检测，并与常规单重PCR进行比较，对该GeXP多重检测体系的临床应用进行评价。【结果】基于GeXP系统的单重PCR检测体系和GeXP多重PCR检测体系均能扩增出特异性片段，验证了引物的可行性、GeXP多重检测体系的特异性和准确性；GeXP多重检测体系的单一病原模板灵敏度分析结果显示，多重检测体系对单一病原体检测的下限均为10¹拷贝/µL；GeXP多重检测体系在8种病原体同时检测的灵敏度分析结果显示，多重检测体系可在10³拷贝/µL水平可同时检测到8种病原体；比较GeXP多重PCR检测方法和常规PCR方法对临床样品的检测结果，两种检测方法的检测结果相符。【结论】成功建立了基于GeXP系统的多重PCR检测体系，可以同时检测8种猪呼吸道和繁殖障碍性疾病病原体；本研究建立的同时鉴别8种猪呼吸道和繁殖障碍性疾病病原体的GeXP检测方法具有高通量、特异性强和灵敏度高的特点，为猪病毒性呼吸道和繁殖障碍性疾病的分子诊断提供了新型的检测方法。

关键词: 猪呼吸道和繁殖障碍传染病, GenomeLab 基因表达平台（GeXP）, 多重PCR, 通用引物, 特异性嵌合引物

Abstract: 【Objective】A GeXP-multiplex PCR assay was developed to simultaneously detect eight swine reproductive and respiratory viruses, including reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus 2 (PCV2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). 【Method】Nine pairs of specific primers were designed based on the conserved sequences of eight pathogens available in the GenBank database. Each gene-specific primer was fused at the 5’ end of a universal sequence to generate 9 pairs of specific chimeric primers. The GeXP-single PCR assay was performed using a single cDNA/DNA template to determine the feasibility of the primers. The GeXP-mutiplex PCR assay was performed using a single cDNA/DNA template and 9 pairs of the specific chimeric primers to determine the specificity and accuracy of the GeXP-mutiplex PCR assay. Serial dilution from 103 copies/μL to 10 copies/μL of in vitro transcripted RNA of target genes (SIV-H1, SIV-H3, PRRSV, CSFV and JEV) and plasmid of PCV-2, PPV and PRV were used to examine the single template sensitivity of GeXP multiplex PCR assay; Serial dilution from 104 copies/μL to 10 copies/μl of all viral targets were used to examine the sensitivity of eight pathogens of GeXP multiplex PCR assay. The GeXP assay was evaluated using 23 clinical specimens and compared with the single PCR assay. 【Result】The corresponding specific fragments of genes were amplified by the GeXP -single PCR assay and the GeXP-mutiplex PCR assay. The results confirmed the feasibility of the primers and the specificity and accuracy of the GeXP assay. The GeXP assay achieved a sensitivity of 10 copies/µL for a single pathogen. The sensitivity of the GeXP assay to detect the eight pathogens simultaneously was 103 copies/µL. The GeXP assay successfully detected twenty-three clinical samples and the results of the GeXP assay were consistent with the results by the single PCR assay.【Conclusion】The GeXP assay was established successfully for simultaneously detecting eight swine reproductive and respiratory pathogens; These results showed that the GeXP assay is a specific, sensitive and high-throughput test to detect swine reproductive and respiratory pathogens. This assay provides a new test for the simultaneous differentiation of eight swine reproductive and respiratory diseases.

Key words: swine reproductive and respiratory diseases, genomeLab gene expression profiler (GeXP), multiplex PCR, universal primers, specific chimeric primers

张民秀，谢芝勋，邓显文，谢志勤，谢丽基，黄莉，黄娇玲. 8种猪呼吸道和繁殖障碍病病原体GeXP检测方法的建立[J]. 中国农业科学, 2015, 48(24): 4996-5006.

ZHANG Min-xiu, XIE Zhi-xun, DENG Xian-wen, XIE Zhi-qin, XIE Li-ji, HUANG Li, HUANG Jiao-ling . Establishment of a GeXP Analyser-Based Multiplex PCR Assay for Detection of Eight Reproductive and Respiratory Swine Pathogens[J]. Scientia Agricultura Sinica, 2015, 48(24): 4996-5006.

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