Development And Preliminary Application Of Loop Mediated Isothermal Amplification (LAMP) For The Detection Of Avian Leukosis Virus Subgroup A And B

Avian leukosis is induced by avian leukosis viruses (ALV), which belongs to genus Alpharetrovirus within the retroviridae family. And it is characterized by neoplastic diseases and immunosuppressive problems in the poultry industry worldwide. ALV can be classified into several subgroups according to the virus neutralization test, viral interference test, and sequence analysis of the envelope protein gene. Chicken ALV, for example, includes exogenous subgroups A, B, C, D, and J and endogenous subgroup E. Currently, the vaccination is not recommended to control this disease, and the best way to eradicate the virus is elimination the infected chickens. Thus, it is important to develop a feasible tool used in detection. Nowadays, the molecular detection tools such as PCR or real-time PCR play an important role in the pathogen identification. Although PCR or real-time PCR are sensitive methods, they require skilled operator and precision equipment which can be expensive and not generally available in clinical utilization. It is necessary and important to develop a rapid, accurate, sensitive and clinical feasible method to detect the pathogen in situ.This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for distinguishing avian leukosis virus (ALV) subgroup A and subgroup B from other subgroups of the virus. On the basis of the results of sequence comparison and the sequence characteristics of ALV subgroups, two LAMP methods were designed differently to target the gp85 segment for detection of ALV-A and ALV-B. Under optimal reaction conditions, ALV-A and ALV-B LAMP produced neither cross-reactions with other major subgroups (including subgroups J, C, and E) nor nonspecific reactions with other common avian infectious diseases. A sensitivity test of ALV-A LAMP showed that this method can detect 20 copies of proviral nucleic acid sequence within 45 min, which is 100 times more sensitive than the conventional polymerase chain reaction (PCR). A sensitivity test of ALV-B LAMP showed that this method can detect 4×103 copies of proviral nucleic acid sequence within 80 min, it was as sensitive as the conventional polymerase chain reaction (PCR). This method can detect subgroup A and B virus rapidly and the results can be observed easily based on color changes with naked eyes under natural light through adding FDR. The whole reaction process can be performed without opening the lid of the reaction tube, which reduces the possibility of contamination greatly and simplifies the detection process, indicating the considerable potential of this method for in situ application in the future.To sum up, the research has successfully established ALV-A and ALV-B examination, and this technology provides theoretical basis for the future research.