Establishment and verification of a TaqMan one-step real-time fluorescence quantitative PCR method for Coxsackievirus B4
10.13200/j.cnki.cjb.004428
- VernacularTitle:柯萨奇病毒B组4型TaqMan探针一步法实时荧光定量PCR检测方法的建立及验证
- Author:
GUO Wei
- Publication Type:Journal Article
- Keywords:
Coxsackievirus B4(CVB4);
TaqMan probe;
Real-time fluorescence quantitative PCR(RT-qPCR)
- From:
Chinese Journal of Biologicals
2025;28(02):167-171
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and verify a one-step real-time fluorescence quantitative PCR(RT-qPCR) method for the detection of Coxsackievirus B4(CVB4) with TaqMan probe, in order to provide a reliable method for rapid quantitative detection of CVB4. Methods According to the more conserved segment of VP1 sequence of CVB4, primers and probes were designed. The positive RNA standard was obtained by in vitro transcription and the standard curve was obtained. The TaqMan probe one-step RT-qPCR method for CVB4 was established and verified for the sensitivity, linear range, reproducibility and specificity. Sixteen clinical samples were detected by using the established method. Results The sensitivity of the method was 10~3 copies/μL. In the concentration range of 10~3-10~(10) copies/μL, the positive RNA standard showed a good linear y x R~2CV relationship with relative fluo rescence unit(RFU),and the line a requation was:=-3.742+48.651,= 0. 998. The of reproducibility verification was less than 2%. The method had no cross-reactivity with other serotypes of coxsackievirus B(CVB2, CVB3, CVB5, CVB6), enterovirus 71(EV71), Coxsackievirus A8, A10 and A12(CVA8, CVA10, CVA12), as well as echovirus 11(E11). Twelve of the 16 clinical samples were positive for CVB4. Conclusion The TaqMan one-step RT-qPCR method developed in this experiment has good sensitivity, specificity and reproducibility, and can be used for the detection of CVB4 clinical samples.
