| Riemerella anatipestifer (RA) infection is a serious world wide problem o f natural waterfowl and, domestic ducks, turkeys and other birds. This has caused high morbidity, mortality, and great economic losses in the poultry industry. Traditional identification methods of RA are laborious and require several days to complete. Various existing test methods, such as the fluorescent antibody technique, and immunohistochemistry methods, have failed to extensive use due to restrictions in serotype differences. Molecular diagnostic technique has allowed the development of novel and powerful tools that can be applied to detection of microbiological agents in clinical conditions, in this study,we establish three molecular diagnostic targeting the gyrB gene for rapid detection of Riemerella anatipestifer.According to gyrB gene sequence from Riemerella anatipestifer,a pair of primer was designed and synthesized. A fragment of194bp was only detected in the RA isolates, while others were negative,which comfimed that the primer and PCR had the high specificity.As little as cultural liquid was16CFU/mL was detectable by this method.Compare of16SrRNA sequence based PCR method,Biolog bacterial Identification system used in detection and identification of suspicious isolates of RA in clinical. The results showed that the use of the gyrB sequences based PCR was exactly consistent with the Biolog identification system, and had better specificity.A set of four primers containing two outer and two inner was designed based on the highly conservative region of RA gyrB gene sequence which has been published on the Genbank. time and temperature were conditions for detection of RA were ptimized for63℃for60min. LAMP products could be judged with agar gel or naked eye after addition of SYBR Green Ⅰ. from the results,both methods showed the equivalent detection threshold The detection of RA using LAMP found to be10-fold more sensitive than that by polymerase chain reaction.,There were no cross reaction with Non-RA strains and the specificity of the assay was comfirm by restriction enzyme digestion of LAMP product. The experiment results show that we developed a rapid, sensitive and specific identification method for detection RA.In this study,we according gyrB gene-specific DNA sequences of RA,The first design a conventional PCR Primers(P1, P2), we used primers P1, P2amplified products were sequenced,the results in GenBank (http://www.ncbi.nlm.gov/BLAST/) are compared.and design187bp FQ-PCR primers(P3, P4)on the basis of sequencing results.The equation of the assay was Y=-3.449X+34.602,and correlation coefficient was0.999and PCR efficiency of crying reached up to95.0%.There was an excel-lent linear relation during the DNA concentration from6×101-6×107copies/μL,and the variation coefficient was less than3%. sensitivity was6×101copies/μL。 However, standard PCR can only be detected6×103copies/μL, The results demonstrating that FQ-PCR is more than100time sensitive than the standard PCR The specificity test showed that only RA strains produced positive reations,no cross-reaction was detected against other avian disease.Through comparison of the three of molecular biologica methods on clinical strainsdetected results show:The three methods can be fast,accurate and specific dentification of the RA isolates.But the sensitivity of FQ-PCR and LAMP are more superior than conventional PCR.However,PCR and FQ-PCR operation requires high-quality training of technical staff and time-consuming. LAMP method does not require expensive equipment.The only equipment needed for the LAMP reaction is a regular laboratory water bath or a heating block that can provide a constant temperature of60-65℃.Moreover,LAMP products can be simply judged with visual inspection by the color change of a mixture with SYBR Green1or a white turbidity of magnesium pyrophosphate.Therefore,LAMP assay is expected to become the effective way for rapid detection of RA. |
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