Pan Xiaoming, Zhang Yanfang, Sha Xuejiao, Wang Jing, Li Jing, Dong Ping, Liang Xingguo
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, P.R. China.
School of Life Sciences, Inner Mongolia University, Hohhot 010000, P.R. China.
J Microbiol Biotechnol. 2017 Mar 28;27(3):471-479. doi: 10.4014/jmb.1606.06053.
White spot syndrome virus (WSSV) is a major threat to the shrimp farming industry and so far there is no effective therapy for it, and thus early diagnostic of WSSV is of great importance. However, at the early stage of infection, the extremely low-abundance of WSSV DNA challenges the detection sensitivity and accuracy of PCR. To effectively detect low-abundance WSSV, here we developed a pre-amplification PCR (pre-amp PCR) method to amplify trace amounts of WSSV DNA from massive background genomic DNA. Combining with normal specific PCR, 10 copies of target WSSV genes were detected from ~10 magnitude of backgrounds. In particular, multiple target genes were able to be balanced amplified with similar efficiency due to the usage of the universal primer. The efficiency of the pre-amp PCR was validated by nested-PCR and quantitative PCR, and pre-amp PCR showed higher efficiency than nested-PCR when multiple targets were detected. The developed method is particularly suitable for the super early diagnosis of WSSV, and has potential to be applied in other low-abundance sample detection cases.
白斑综合征病毒（WSSV）是对虾养殖业的主要威胁，目前尚无有效的治疗方法，因此WSSV的早期诊断至关重要。然而，在感染早期，WSSV DNA的极低丰度对PCR检测的灵敏度和准确性提出了挑战。为了有效检测低丰度的WSSV，我们开发了一种预扩增PCR（pre-amp PCR）方法，从大量背景基因组DNA中扩增痕量的WSSV DNA。与常规特异性PCR相结合，在约10倍背景下检测到10个拷贝的目标WSSV基因。特别是，由于使用了通用引物，多个目标基因能够以相似的效率进行平衡扩增。通过巢式PCR和定量PCR验证了pre-amp PCR的效率，在检测多个目标时，pre-amp PCR显示出比巢式PCR更高的效率。所开发的方法特别适用于WSSV的超早期诊断，并且有潜力应用于其他低丰度样品检测案例。
