基于直扩RT-PCR技术的寨卡病毒快速检测方法的建立  

Establishment of rapid detection method for zika virus based on direct amplification RT-PCR technique

作　　者：李浪 古莉冰 朱丽 何建安 叶颖 张然 李华文 李福缘 顾大勇 LI Lang;GU Libing;ZHU Li;HE Jianan;YE Ying;ZHANG Ran;LI Huawen;LI Fuyuan;GU Dayong(School of Public Health,Guangdong Medical University,Dongguan,Guangdong 523808,China;Shenzhen Academy of Inspection and Quarantine,Shenzhen,Guangdong 518033,China;Shenzhen Customs District of the People′s Republic of China,Shenzhen,Guangdong 518026,China;Shenzhen International Travel Healthcare Center/Shenzhen Customs Port Clinic,Shenzhen,Guangdong 518045,China;Community Health Service Management Center,Baoan Hospital of TCM,Shenzhen,Guangdong 518133,China)

机构地区：[1]广东医科大学公共卫生学院,广东东莞523808 [2]深圳市检验检疫科学研究院,广东深圳518033 [3]中华人民共和国深圳海关,广东深圳518026 [4]深圳国际旅行卫生保健中心/深圳海关口岸门诊部,广东深圳518045 [5]深圳市宝安区中医院社区健康服务管理中心,广东深圳518133

出　　处：《国际检验医学杂志》2024年第3期358-364,共7页International Journal of Laboratory Medicine

基　　金：广东省科技计划资助项目(2016A020219005);2021年海关总署科研项目(2021HK143)。

摘　　要：目的建立基于直扩实时荧光定量逆转录聚合酶链反应(RT-PCR)技术的寨卡病毒快速检测方法。方法采用特殊功能的DNA聚合酶,以及优选PCR增强剂,以此建立直扩RT-PCR技术的寨卡病毒快速检测5种样本(全血、血清、唾液、咽拭子和尿)的方法。结果5种样本检测限分别为血清103 PFU/mL,尿、咽拭子和唾液102 PFU/mL,全血104 PFU/mL,标准曲线的拟合优度的可决系数均在0.98以上,扩增效率均在90%~110%;寨卡病毒核酸成功扩增,非寨卡病毒核酸均未能扩增;尿、全血和唾液样本的重复性实验中106 PFU/mL和102 PFU/mL两个浓度的6个重复Ct值的变异系数均<5%。该研究建立的直扩RT-PCR技术的寨卡病毒检测方法与常规RT-PCR技术的检测结果一致,8个寨卡病毒样本,均只检测出2个血清样本,其余62个非寨卡病毒样本及12个阴性样本均未得到扩增。结论成功建立基于直扩RT-PCR技术的寨卡病毒快速检测方法,该方法简便快捷且灵敏度高、特异度强。Objective To establish a rapid detection method for zika virus based on direct amplification real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)technique.Methods A direct amplification RT-PCR technique for the rapid detection of zika virus in 5 samples(whole blood,serum,saliva,throat swab and urine)was established by using a special function DNA polymerase and a preferred PCR enhancer.Results The detection limits of the 5 samples were 103 PFU/mL in serum,102 PFU/mL in urine,throat swab,and saliva,and 104 PFU/mL in whole blood.The coefficient of goodness-fit of standard curves was above 0.98,and the amplification efficiency was 90%-110%.Zika virus nucleic acid was successfully amplified,but non-zika virus nucleic acid was not amplified.Based on the repeatable detection of samples from urine,whole blood,and saliva,the variation coefficient of 6 repeated Ct values at 106 PFU/mL and 102 PFU/mL concentrations were all<5%.The zika virus detection method established by the direct amplification RT-PCR technique was consistent with the detection results of conventional RT-PCR technique.Only two serum samples were detected in eight zika virus samples,and the remaining 62 non-zika virus samples and 12 negative samples were not amplified.Conclusion A rapid detection method for zika virus based on direct amplification RT-PCR technique is successfully established.The method is simple,rapid,sensitive and specific.

关 键 词：寨卡病毒 直扩实时荧光定量逆转录聚合酶链反应技术 DNA聚合酶 

分 类 号：R446.5[医药卫生—诊断学]