六种猪呼吸道RNA病毒MALDI-TOF MS同步检测体系的构建

A MALDI-TOF MS system for simultaneous detection of six RNA viruses causing porcine respiratory diseases

摘要:

【目的】 建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)、猪瘟病毒(classical swine fever virus, CSFV)、口蹄疫病毒(foot-and-mouth disease virus, FMDV)和猪流感病毒(swine influenza virus, SIV)的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)多目标检测方法,同时对SIV进行通用型、H1型及H3型分型检测。【方法】 本研究根据6种病原体基因的保守序列,设计了6对加标引物及对应的延伸探针并进行单反应试验。通过体系优化引物浓度和反应条件,以及方法特异性、重复性及灵敏度分析,使用MALDI-TOF MS检测方法及荧光定量PCR方法分别对临床样本和猪源产制品进行检测,并对结果进行对比验证。【结果】 质谱结果显示,6种产物峰仅在靶标病毒对应的产物位置出现峰值,其他常见猪病病原在质谱图像中均未出现干扰,表明该体系的特异性良好。体系中每种病毒在高、中、低浓度时的批内阳性符合率均为100%,批间符合率均 ≥ 96.7%,表明该体系的重复性良好。灵敏度分析结果显示,体系中各病毒最低检测限在6.73–21.25拷贝/μL范围。应用MALDI-TOF MS多重检测方法对124份组织、饲料及猪肉样品进行检测,检出PRRSV阳性样本9份,SIV阳性样本3份(其中SIV-H1阳性样本2份,SIV-H3阳性样本1份)。将以上结果与荧光定量PCR方法分析结果进行对比验证,2种方法对各病原体检测结果的总符合率可达到99.2%–100%。【结论】 本研究建立的6种猪呼吸道RNA病毒MALDI-TOF MS同步检测方法为相关疫病监测和快速鉴别,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 multi-target method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), and swine influenza virus (SIV), and the detection of the general type, H1 type and H3 type of SIV. [Methods] We designed six pairs of primers and corresponding extension probes according to the conserved sequences of six pathogens and conducted single reaction tests. The primer concentrations and the reaction conditions were optimized for the established method. After examination of the specificity, repeatability, and sensitivity, the MALDI-TOF MS method was used to detect clinical samples and pig-derived products, and the results were compared with those obtained by fluorescence quantitative PCR. [Results] The established method only generated peaks corresponding to the positions of the target viruses, and other common pathogens causing swine diseases did not appear in the mass spectra, which indicated that the system had good specificity. The in-batch positive coincidence rate of each virus at high, medium, and low concentrations was 100%, and the inter-batch coincidence rate was ≥ 96.7%, indicating that the system had good repeatability. The minimum limit of detection for each virus in the system varied within 6.73–21.25 copies/μL. We then used the MALDI-TOF MS method to detect 124 tissue, feed, and pork samples. A total of 12 positive samples were detected, including nine PRRSV positive samples and three SIV positive samples (two SIV-H1 positive samples and one SIV-H3 positive sample). We then compared the above results with the results from fluorescence quantitative PCR, and found that the total coincidence rate of the two methods for detection of each pathogen was 99.2%–100%. [Conclusion] A method for the simultaneous detection of six RNA viruses causing porcine respiratory diseases was established based on MALDI-TOF MS. This sensitive and specific high-throughput multi-target detection method facilitates the surveillance and rapid identification of related diseases in the import and export animal quarantine.