The Application Of Loop-mediated Isothermal Amplification In The Diagnosis Of Brucella And Human Papillomavirus

Loop-mediated isothermal amplification(LAMP)is a process of nucleic acid amplification under temperature condition,which has the advantages of being rapid,portable,sensitive and specific.This study intends to establish improved ring mediated isothermal amplification(loop-mediated isothermal amplification,LAMP)technology,the traditional LAMP technology on the basis of the results of determination,the reaction system and nucleic acid extraction method were improved,and applied to the rapid visual detection undulant and human papillomavirus(HPV)two kinds of common pathogens,and try to promote applied to infection or grassroots medical institutions where the original site,with each one,for example,bacteria and viruses to complete part 2 experimental study.Part ? Establishment of a modified loop-mediated isothermal amplification method for direct detection of Brucellosis in patient and sheep blood samplesObjective: To establish a modified loop-mediated isothermal amplification(LAMP)technique for the detection of Brucellosis in blood samples from patients and infected sheep.Methods:For brucella Omp25 gene sequence conservative design LAMP amplification primer,optimize the LAMP system,through the gradient test respectively to cultivate undulant cattle,sheep,pigs standard strains and 13 species of gram-negative bacteria as positive and negative control,collect solstice on May 10,2018 on November 1,2019 diagnosed with blue patients blood samples of 104 cases healthy people 50 cases of blood sample,collect collect farms in hebei,gansu and Inner Mongolia,Hong Kong and sheep blood specimens were suspected to have contracted undulant 200 cases as test sample,boiling method was applied to extract the above strains and samples of DNA.Positive and negative control strains were detected by optimized LAMP method to verify their specificity.LAMP system and Q-PCR were used to detect 10-104 copies/ L isolates in gradient dilution and compare the sensitivity of the two methods.Clinical patient specimens were detected by RBPT,LAMP and PCR respectively,while sheep blood specimens were detected by LAMP and PCR.Finally,SPSS 21.0 was used to calculate Kappa values of different detection results,and the consistency of the three detection methods was compared to verify the specificity and sensitivity of LAMP method in different specimens.Results: Optimized by LAMP system,it was found that the amplification efficiency was highest when the final concentrations of Mg SO4 and betaine were 8 m M and 0.8 m M,respectively.No non-specific amplification was observed in LAMP specificity experiment,indicating its good specificity.LAMP detected 10 copies/ L of the standard strain of Brucella,which was one order of magnitude higher than that of Q-PCR.LAMP showed good sensitivity and specificity in patients and sheep blood samples(98.70% and 100%,respectively).(Kappa > 0.7),with good consistency(P > 0.05).Conclusion: THE sensitivity of LAMP was superior to PCR,and the detection results of different types of specimens were the same as other methods.However,due to its simpler operation and lower operational risk,LAMP was considered to be more suitable for basic or field detection with poor conditions.Part ?? Establishment of a modified loop-mediated isothermal amplification technique for direct detection of human papillomavirusObjective: To apply improved loop-mediated isothermal amplification(LAMP)visualization for detection of human papillomavirus(HPV)subtypes 16,18,52,and 58,and explore the application prospect of visual LAMP technology in grassroots or primary hospitals.Methods: First,collecting the chengde medical college affiliated hospital clinical laboratory identification of HPV16,18,52,58 specimens of cervical exfoliated cells each single subtype infection in 1 case for system set up,collected in October 2018 and August 2018-Yu Chengde medical school affiliated hospital of suspected of HPV infection in patients with cervical exfoliated cytology specimens,a total of 452 cases for samples.Then,LAMP primers were designed according to E6 and E7 regions of HPV16,18,52 and 58 subtypes,and LAMP system was optimized by gradient test.The specificity was verified by template exchange experiment,the sensitivity of the template was analyzed by detecting gradient dilution,and the accuracy of the visualization results was verified by adding calcitonin to the system and comparing the color development results with electrophoretic images.Finally,PCR was used for qualitative detection of all clinical specimens,and LAMP and PCR were used for typing of HPV positive specimens.SPSS 21.0 software was used to calculate the Kappa value of LAMP and PCR typing results,and the consistency analysis was conducted to compare the statistical differences between the two methods.Results: LAMP system with better amplification effect was established through optimization experiment.After optimization,it showed good specificity without non-specific amplification,and the detection limit of HPV16,18,52,and 58 subtypes was 100,103,100,103 copies/ L respectively(the detection limit of HPV16 and 52 subtypes was one order of magnitude lower than normal PCR).The results obtained by adding calcitonin were comparable to those obtained by electrophoresis.Based on clinical samples,specimens of 452 cases,163 cases of HPV positive specimens,accounted for 36.1%,including LAMP method to detect the 34 cases of HPV16 positive(22.1%),17 cases HPV58 positive(10.4%),18 cases HPV52 positive(11.0%)and 6 cases of HPV18 positive(3.7%),the LAMP with PCR classification statistical differences between test results is not obvious(Kappa = 0.854,P > 0.05),the consistency of the two methods is better.Conclusion: LAMP has good specificity,and the detection lower limit of HPV16 and 52 subtypes is one order of magnitude higher than ordinary PCR,and the sensitivity is better than ordinary PCR.In terms of clinical specimen detection,LAMP and PCR were in good agreement,but LAMP could detect unpurified HPV virus,and the detection results could be determined directly by naked eyes.The operation process was simpler,and it was more suitable for basic or field detection with poor conditions.