Establishment And Application Of A Loop-mediated Isothermal Amplification Method For Detection Of Duck Hepatitis Virus Type Ⅰ

Duck viral hepatitis is caused by duck hepatitis virus (DHV) which is an acute, highly lethal infectious disease.It seriously restricts the duck industry development. Duck hepatitis virus consist of three types.Duck hepatitis virus type Ⅰ (DHV-Ⅰ) belong to the small RNA virus families, with no capsule.Loop-mediated isothermal amplification (LAMP) technology is a new type of nucleic acid amplification technology. With the special primers and DNA polymerase, nucleic acid amplification can be achieved under isothermal conditions.It has the advantages of simple operation, low cost, fast speed. The technology has been used in the detection and diagnosis of many diseases application.So that, a LAMP method for detection of DHV-Ⅰ was established, and it was successfully applied in detection of duck cloacal duck hepatitis virus. It provides a new method for the diagnosis of this disease.First of all, collect the gene sequence of DHV-Ⅰ from GenBank. The 3D gene sequences is the most conservative after biological information analysis. So it was chosen as the target sequence. Multiple sets of LAMP primers were designed by online primer software. Select the primer set which can be used for detection by pre-experiment.Secondly, through the optimization of the reaction system (Mg2+ concentration, betaine concentration, dNTPs concentration, Bst DNA polymerase concentration, primers concentration) and conditions (temperature and time) one by one, a new DHV-Ⅰ LAMP detection method was established. The sensitivity of the method is 10fg, which is 100 times as much as PCR.That is especially good to tissue disease of low virus content material.The nucleic acid of DHV-Ⅰ was used as positive samples, and the nucleic acid of other common duck pathogens (DHV-Ⅱ、DHV-Ⅲ、MDRV、NDV、DPV、GPV、DPV) and DEPC water were detected as negative samples. The results showed that the positive samples had white precipitation, while the negative samples had no change.Amplification reaction in the water bath for 45 min can be completed.And the result can be determined by naked eyes directly.Finally, the LAMP method established in this study is used for detection of DHV-Ⅰ in 160 samples.The positive rate is 27%.It provides the basis for treatment for guiding the development of an immunization program. It lays an important foundation for the development of a kit.