超分支滚环扩增试纸检测对虾黄头病毒

The Detection of Yellow Head Virus by Hyper-Branched Rolling Circle Amplification Test Strip

中文关键词: 对虾黄头病毒  超分支滚环扩增法  锁式探针  检测试纸

英文关键词: Yellow head virus  HRCA  Padlock probe  Test strip

中文摘要:

      依据对虾黄头病毒(Yellow head virus, YHV)的非结构蛋白N基因序列，设计特异的锁式探针(Padlock probe, PLP)、检测探针及引物，建立YHV超分支滚环扩增(Hyper-branched rolling circle amplification, HRCA)检测试纸。灵敏度实验显示，YHV HRCA检测试纸能检测出的最低模板量为101拷贝，是RT-PCR灵敏度的100倍。特异性实验结果表明，该试纸能够特异性地对YHV进行检测。利用该检测试纸对进出口80批次虾样本进行检测，并将检测结果与常规RT-PCR相比较，结果显示，YHV HRCA检测试纸灵敏度方面优于常规RT-PCR方法，且操作简便、结果直观易读。

英文摘要:

      In this study we aimed to develop an economical, visual-friendly, and portable test to detect yellow head virus (YHV) in field. Based on sequence of nonstructural protein N of YHV, a padlock probe (PLP), detection probe, and universal primers were designed. Subsequently the hyper-branched rolling circle amplification (HRCA) assay and the corresponding test strip were developed. The reaction time and temperature were optimized. Padlock probes were linked to the target sequence by T4 DNA ligase at 37℃ for 30 min, and reacted by Bst DNA polymerase large fragments at 61℃ for 30 min. The test strip was then made using the detection probe. The serials diluted reference materials were used to examine the sensitivity of the YHV HRCA test strip, and the result was compared with that of the conventional reverse transcription polymerase chain reaction (RT-PCR). Three primary shrimp viruses including white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, and taura syndrome virus were used for the specificity test of the YHV HRCA test strip. Furthermore, the efficiency of the test strip was verified with 80 patches of 4 shrimp species collected in China and abroad, and the results were also compared with RT-PCR. The test results showed that the detection limit of the HRCA test strip was close to 10 copies/μl, which was 100-fold higher than RT-PCR. It was also shown that the test strip had a satisfying specificity for YHV, and there were no cross-reaction with white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, or taura syndrome virus. The overall YHV test results using the HRCA test strip were identical with RT-PCR, but the former was more convenient, sensitive, and easier to interpret in the field.