传染性非洲猪瘟病毒(ASFV)可引起非洲猪瘟的传播和发病,而灭活的病毒不能。当它们没有分开区分时,检测结果会缺乏真实性,造成不必要的恐慌和检测成本。基于细胞培养的检测技术复杂,高成本,在实践中耗时,不利于传染性ASFV的快速检测。在这项研究中,构建了一种快速诊断感染性ASFV的单叠氮丙啶(PMA)qPCR检测方法。PMA浓度参数,光强度,和照明时间进行了严格的安全验证和比较分析,以进行优化。结果确定PMA预处理ASFV的最佳条件是PMA100μM的终浓度。光强度为40W,光照时间为20分钟,最佳引物探针的目标片段大小为484bp,对感染性ASFV的检测灵敏度为101.28HAD50/mL。此外,该方法创新性地应用于消毒效果的快速评价。当ASFV浓度小于102.28HAD50/mL时,该方法仍然可以有效地评估热灭活效果,含氯消毒剂的评价能力较好,适用浓度可达105.28HAD50/mL。值得一提的是,这种方法不仅可以反映病毒是否灭活,也间接反映了消毒剂对病毒核酸的破坏程度。总之,本研究构建的PMA-qPCR可应用于实验室诊断,消毒效果评价,药物开发,以及其他方面的传染性ASFV,可以为有效预防和控制ASF提供新的技术支持。关键点:•开发了一种用于感染性ASFV的快速检测方法•提供一种用于快速评估含氯消毒剂的消毒效果的新方案•PMA-qPCR可以同时显示病毒的存活状态和核酸的损伤。
Infectious African swine fever virus (ASFV) can cause the spread and morbidity of African swine fever, while the inactivated virus cannot. When they are not distinguished separately, the detection results will lack authenticity and cause unnecessary panic and detection cost. The detection technology based on cell culture is complex, high-cost, and time-consuming in practice, which is not conducive to the rapid detection of infectious ASFV. In this study, a propidium monoazide (
PMA) qPCR detection method for rapid diagnosis of infectious ASFV was constructed. Parameters of
PMA concentration, light intensity, and lighting time were under strict safety verification and comparative analysis for optimization. The results determined that the optimal condition for
PMA to pretreat ASFV was the final concentration of
PMA 100 μM. The light intensity was 40 W, the light duration was 20 min, the target fragment size of the optimal primer probe was 484 bp, and its detection sensitivity for infectious ASFV was 101.28 HAD50/mL. In addition, the method was innovatively applied to the rapid evaluation of disinfection effect. When ASFV concentration was less than 102.28 HAD50/mL, the method could still be effective for the evaluation of thermal inactivation effect, and the evaluation ability of chlorine-containing disinfectants was better, and the applicable concentration could reach 105.28 HAD50/mL. It is worth mentioning that this method can not only reflect whether the virus is inactivated, but also indirectly reflect the degree of damage to viral nucleic acid caused by disinfectants. In conclusion, the
PMA-qPCR constructed in this study can be applied to laboratory diagnosis, disinfection effect evaluation, drug development, and other aspects of infectious ASFV and can provide new technical support for effective prevention and control of ASF. KEY POINTS: • A rapid detection method for infectious ASFV was developed • Provide a new scheme for rapid evaluation of disinfection effect of chlorine-containing disinfectants • PMA-qPCR can simultaneously show the survival status of the virus and the damage of nucleic acid.