基于基质辅助激光解吸/电离飞行时间质谱的猪呼吸道病毒多目标鉴定方法的建立和应用

Establishment and application of a multi-target detection method for porcine respiratory viruses based on MALDI-TOF MS

摘要:

【目的】建立能高效同步鉴定猪伪狂犬病毒(porcine pseudorabies virus, PRV)、猪圆环病毒2型(porcine circovirus 2, PCV-2)和3型(porcine circovirus 3, PCV-3)、非洲猪瘟病毒(African swine fever virus, ASFV)以及猪博卡病毒1型(porcine bocavirus group 1, PBoV-G1)、2型(porcine bocavirus group 2, PBoV-G2)和3型(porcine bocavirus group 3, PBoV-G3)等呼吸道病毒的核酸基质辅助激光解吸/电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS)高通量多目标检测技术。【方法】根据7种病原体基因的保守序列，分别设计不同病原的引物及对应的单碱基延伸探针，通过引物浓度和反应条件优化，方法特异性、敏感性和稳定性分析，以及临床样本和猪源产制品的检测验证，建立常见猪呼吸道DNA病毒的MALDI-TOF MS多目标检测体系。【结果】质谱分析显示，多目标检测体系的7种靶标产物峰只在特定病毒阳性样品检测时产生，与其他病原体检测无交叉反应，表明该方法对7种靶标病毒检测特异性良好。重复性试验结果分析显示，体系中每种病毒在高、中、低浓度时批内阳性符合率均≥98.0%，批间均≥98.3%，表明该方法具有较高的稳定性。体系中7种病原体每种病毒最低检测限在8.65-26.27拷贝/μL之间，与荧光PCR (real-time fluorescence quantitative PCR, RT-qPCR)检测方法相当。采用MALDI-TOF MS多重检测方法对100份组织、饲料和猪肉样品进行检测应用，检出2种及以上混合感染样品39份，其中5份样本同步检出5种病原体阳性；对8份ASFV-p72假病毒人工污染样品进行验证，均可检出ASFV阳性。将以上样本检测应用结果与荧光PCR方法进行比对验证，2种方法对于不同病原体检测结果的符合率高达94.4%-100%。【结论】本研究建立的基于MALDI-TOF MS的猪呼吸道常见DNA病毒多重检测方法为猪群相关疫病快速监测和鉴别诊断，以及便利化进出口动物检疫等提供了一种新的敏感、特异的高通量多目标检测技术。

Abstract:

[Objective] To establish a high-throughput multi-target technique based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of porcine pseudorabies virus (PRV), porcine circovirus types 2 (PCV-2) and 3 (PCV-3), African swine fever virus (ASFV), and porcine bocavirus group 1 (PBoV-G1), group 2 (PBoV-G2) and group 3 (PBoV-G3). [Methods] The primers for different pathogens and corresponding single-base extension probes were designed based on the conserved sequences in the genes of the seven pathogens. After the optimization of primer concentrations and reaction conditions, the specificity, sensitivity, and repeatability of the method were analyzed. Finally, this MALDI-TOF MS method was used to detect the pathogens in clinical samples and porcine products. [Results] The established method only produced target peaks in the detection of specific virus-positive samples, and there was no cross-reaction with other pathogens, which indicated that the method had good specificity for the detection of the seven target viruses. The repeatability test results showed that the positive coincidence rate of each virus in high, medium, and low concentrations was ≥98.0% within batches and ≥98.3% between batches, indicating that the method had high repeatability. The low limits of detection of the established method for the seven pathogens in the system were within the range of 8.65-26.27 copies/μL, which was comparable to that of the real-time fluorescence quantitative PCR (RT-qPCR). The established MALDI-TOF MS method was used to detect 100 tissue, feed, and pork samples, in which 39 samples were detected with two or more mixed infections, including five samples simultaneously positive for five pathogens. In addition, eight samples of artificial contamination with recombinant adenovirus carrying ASFV-p72 gene were tested, which showed ASFV-positive results. The coincidence rates of the results obtained by MALDI-TOF MS and RT-qPCR for the detection of different pathogens were as high as 94.4%-100%. [Conclusion] The method established based on MALDI-TOF MS for the detection of multiple porcine respiratory DNA viruses in this study provides a sensitive, specific, high-throughput and multi-target detection technique for rapid surveillance and differential diagnosis of porcine diseases, facilitating the import and export animal quarantine.