| Infectious bronchitis（IB）is a serious,highly contagious respiratory disease caused by infectious bronchitis virus（IBV）.The disease is currently prevalent worldwide,mainly infecting 1-4 weeks old young chickens,which is one of the important diseases affecting the development of poultry industry.Vaccine immunization is the main measure to prevent and control IB.Although a variety of attenuated live vaccines have been applied to chickens,immunized flocks still frequently develop IB,mainly because the IBV genome is highly susceptible to mutation,leading to the occurring of new genotypes and even new serotypes.However,the cross-protection between different genotypes/serotypes is limited,so immune failures often occur,which brings great difficulties to the prevention and control of the disease.Therefore,the rapid and accurate diagnosis of IBV is very important,and rapid identification of IBV genotypes is the main link to effectively prevent the disease.In view of this,it is very necessary to develop sensitive,specific,rapid,and simple IBV diagnostic techniques and timely identification of IBV genotypes.Therefore,the development and preliminary application of rapid diagnostic techniques of Nano-PCR and type-specific LAMP for IBV were carried out in this study.Firstly,according to the conserved region of the 3’non-coding region of IBV,a pair of specific primers were designed and synthesized to develop a Nano-PCR detection method for IBV,and the specificity,sensitivity and reproducibility of the Nano-PCR method were evaluated.Also,the developed Nano-PCR method was applied to detect samples of the clinical cases and the challenged birds.The results showed that the developed Nano-PCR method was specific and a band of about 346bp was only detected from the nucleic acid sample of IBV,but not from avian metapneumovirus,infectious bursal disease virus,Marek’s disease virus,Newcastle disease virus,avian leucosis virus,avian influenza virus H9 subtype,infectious laryngotracheitis virus,duck hepatitis virus and other pathogens.Sensitivity tests showed that the minimum detection concentration of Nano-PCR was 1.01×10-4ng/μL,and that was 10 times of the conventional PCR.The results of the reproducibility test showed that the extracted IBV nucleic acid virus can be detected repeatedly three times.The application test showed that the positive rate of the clinical and challenged samples were 50%（10/20）and 60%（30/50）,respectively,and the coincidence rate with the conventional PCR method was100%.Secondly,according to the S1 gene sequences of the IBV Mass and 4/91strains in Gen Bank,a set of Mass and 4/91 type specific loop-mediated isothermal amplification（LAMP）primers were designed.After optimizing the LAMP reaction system and reaction conditions,the Mass and 4/91 type specific LAMP diagnostic methods were developed,and the specificity,sensitivity and reproducibility of the type-specific LAMP were also determined.In addition,the developed type-specific LAMP was applied to the clinical samples.The results showed that both Mass and4/91 type specific LAMP diagnostic methods could complete the amplification of corresponding IBV genotypes in 60 minutes,without cross-reactivity with other genotypes of IBV,Newcastle disease virus,infectious bursal disease virus,Marek’s disease virus,avian leucosis virus.Sensitivity tests showed that the minimum detection concentration of Mass-specific LAMP was 1×10-4 ng/μL,being 100 times that of conventional PCR,and the minimum detection concentration of4/91-specific LAMP was 9×10-4 ng/μL,being 10 times that of conventional PCR.The results of repeated above tests for three times were same.Detection of 20 IBV strains saved in our laboratory by the developed IBV type-specific LAMP diagnostic method was performed.The results showed that 5 out 20 strains were4/91 type IBVs,6 were Mass type IBVs,which were consistent with the results of genotype based on the S1 gene of these strains by our research group.The IBV Nano-PCR detection method was firstly developed in the present study and has higher sensitivity and good specificity,providing a new technical means for clinical diagnosis,pathogen detection and epidemiological investigation of IBV.At the same time,the Mass and 4/91 type specific LAMP detection methods were developed and have higher sensitivity and good specificity,which is of great significance in effective disease control,pathogen research,and epidemiological investigation and so on.Moreover,these methods have the advantages of low cost,simple operation and no need special equipments,so they are worth popularizing and applying in chicken farms and basic business departments. |
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