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

Abstract:

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.