Research And Application Of Detection Of Rifampicin Resistant Mycobacterium Tuberculosis Based On Isothermal Amplification Technique

Objective:Tuberculosis is a chronic disease caused by Mycobacterium tuberculosis,and could occure in different organs of the body,mainly in lung.Mycobacterium tuberculosis has a long cycle of treatment and is prone to drug resistance.Multidrugresistant TB and extensively drug-resistant TB bring great challenge to the diagnosis and treatment of Mycobacterium tuberculosis.The appearance of drug-resistant TB is due to the mutations in related genes,and the test for these mutations could determined whether Mycobacterium tuberculosis is drug-resistance or not.The rifampicin resistant TB is caused by the gene mutation of rpoB,and previous studies have proved 90%of rifampicin resistant isolates are also isoniazid resistant,so rifampicin resistance could be used as a marker for the detection of multidrug resistant tuberculosis.Currently,the molecular technologies for the diagnosis of Mycobacterium tuberculosis are based on sequence,the linear probe hybridization and PCR.However,the steps of above technologies are complicated and they need expensive instruments and professionals,bringing great difficulties for the detection of drug-resistant TB in resource-poor areas.So,It is urgent to develop new detection technology for the diagnosis of drug-resistance TB.With the development of technical methods on nucleotide detection,a variety of methods on isothermal amplification were developed.Unlike PCR detection,isothermal amplification does not need changes of temperature during reaction,thereby reducing the requirement for associated instruments.What’s more,isothermal amplification also has many advances such as high amplification efficiency,lower cost,easy to operate and so on.To date,Loop-mediated amplification(LAMP)and rolling circle amplification(RCA)are more reliable methods in the aspects of isothermal amplification.LAMP could amplify nucleotide with high efficiency by recognizing 6 regions of targeted nucleic acid sequence using 4 primers(2 inner primers and 2 outer primers).During LAMP,a hairpin structure is formatted via inner and outer primers,then a rapid amplification is on process by inner primers.In addition to bacteria detection,LAMP is also available for SNP detection.Detecting TB using LAMP has been recommended by WHO in 2016.However,to date,there is no report on the detection of drug resistance of TB using LAMP.In this study,aiming at rifampicin resistant TB,we want to expore the possibility of drug resistant TB detection using LAMP,thereby providing a lower cost and more convenient method for the diagnosis of drug resistant TB.RCA is a method which simulate the process of viral genome replication by using targeted microRNA or a long-strand nucleotides as primers to extent circle templete strand.RCA reveals its great potential no matter on basic research or on clinical application such as the detection of microRNAs or SNP.Therefore,in this study,we also performed a systematic research on RCA,in the hope to provide a new method to detect drug resistant TB with a high specificity.Methods:1.The solid culture was used to go on drug susceptibility testing for derterming whether TB is drug-resistant or not.Then,extract the genome of rifampicin-resistant TB,and test its concentration.Gene sequencing was used to determined the mutations in rpoB gene.2.Based on the design principles of PCR primer,primer 5.0 was used to design PCR primers.3.Based on the design principles of LAMP primer,online software(http://primerexplorer.jp/lampv5e/index.html)was used to design LAMP primers.4.LAMP was used to detect the clinical Mycobacterium tuberculosis,and chose the best suitable set of LAMP primers.5.Agarose gel electrophoresis was used to test the production of PCR and LAMP.6.Polyacrylamide gel electrophoresis was used to detect hybridization production of PNA and DNA.7.DNAMAN was used to design padlock probe of RCA and connection sequence.8.RCA was used to amplify the CELI digestion product.Results:1.The results of sequence on rpoB gene in 102 samples of which the TB has rifampicin resistance indicate that 68/102 cases have a mutation on 531 site of rpoB gene,which is the most frequent mutation.In addition,mutation on site 526 and site 516 has the second and the third mutation frequency respectively.2.Different substances have different effects on LAMP non-specific amplification.Pullulan shows an ability to inhibit the non-specific amplification of LAMP significantly,and this inhibitary effect is concentration dependent.DMSO,TMSO and glycerol could slightly release the non-specific amplification from primer hybridization of LAMP,but glycerol could repress the sample amplification.Bovine serum albumin(BSA)and DLdithiothreitol(DTT)promote non-specific amplification from primer hybridization of LAMP.3.The addation of mutation beside the LAMP primer mutation can not distinguish the drug-resistance strains from non-resistance strains,but will reduce the LAMP reaction efficiency.4.The addation of TaqMuts in LAMP amplification system will not distinguish the drugresistant strains from non resistance strains,and the result shows that the collected amplification singal is reduced after the addition of TaqMuts.5.When PNA and DNA hybrid,non-complementary base will exert great effect on their hybridization.The hybridization of PNA and DNA will not be affected by the concentration of saltion,but DTT reduce the possibility of the combination of PNA and DNA.When the DTT concentration exceeds 1 mM,PNA and DNA can not hybrid.6.PNA hybrids with completely complementary DNA could protect the DNA from being degisted by CELI7.CELI digests the hybridization production of PNA and DNA,if the production is completely complementary,a small hybridization fragment of 20 bp will be left.But if there is a mutation,CELI could digest the mismatch,small amount of production or no production will be left.8.RCA amplifies hydrolyzed product by CELI,if there is no mutations,the amplification signal will be got,but if there is a mutation,no signal will be collected.Compared with sequencing,the sensitivity and specificity of the combination of PNA,DNA for detecting SNP are respectively 100%and 95.45%,determining if there is a mutation at 531 code of rpoB gene in Mycobacterium tuberculosis accurately.Conclusion:1.Pullulan could release or completely repress the non-specific amplification from primer hybridization of LAMP.2.In present,LAMP can not distinguish if the clinical isolates are drug-resistance.3.The combination of RCA,PNA and DNA for detecting SNP could success determing that if there are mutations in target genome,offering a new method for SNP detection.And comparing with gene sequencing,this way could detect 531 code of rpoB gene in Mycobacterium tuberculosis with the sensitivity and specificity of 100%,95.45%respectively.