New Methods For Mycobacterium Tuberculosis Detection Based On Nucleic Acid Amplification Techniques

Tuberculosis(TB)is an infectious disease caused by the infectious pathogenic bacterium-Mycobacterium tuberculosis(MTB),and it remains a major public health problem in both developing and developed countries.Currently existing diagnosis methods,such as smear microscopy,culture studies and chest X-radiography,are suffer from certain limitations.For instance,smear microscopy method has the drawbacks of poor sensitivity,disappointed detection rates and low throughput;bacterial cultures requires sophisticated laboratory facilities and weeks to months to complete because of the slow growth of MTB,and chest radiography is limited by the insufficient specificity for immunocompromised and HIV infected patients.The advances in nucleic acid amplification technology(NAAT)triggers the development of new rapid methods for TB diagnosis.These methods have the advantages of rapidity and high sensitivity,however,the using of enzyme for signal amplification and the requirement of sophisticated laboratory instruments and well-trained personnel limit the application of the nucleic acid amplification-based MTB detections.Based on these,new enzyme-free methods based on NAAT were developed for simple,rapid and cost-saving MTB detection.The reseach details are as follow:(1)MTB detection based on strand displacement amplification(SDA)and magnetic beads(MBs).In this text designed two haprin probes the MBs-labled Hp1 and TAMRA-labled Hp2 were used for MTB 16 sDNA detection.In the absence of MTB 16 sDNA,the two probes maintain the loop-stern structure,and the haiprin structure of Hp1 will be opened with the addition of MTB 16 sDNA because of the complementary of MTB 16 sDNA to the stern section of Hp1.Then the opened Hp1 will hybridize with TAMRA-Hp2 to from double strand DNA,and MTB 16 sDNA disassociates and opens another loop-stern structured Hp1.Under the optimal conditions,there is a good linear relationship between the fluorescence and the concentration of MTB 16 sDNA ranging from 0.05 to 150 nM with the correlation coefficient of 0.990(n = 8)and the detection limit of 27 pM(3?/K).Moreover,the MTB 16 sDNA contents in PCR samples were successfully detected with the new method,and the recoveries were 94.4-100.4%,suggesting the high accuracy of the method.(2)MTB detection based on HCR and MBs.HCR is a novel non-enzymatic signal amplification method.Classical HCR reactions include two DNA hairpin probes,which stabilize the neck ring structure when there is no target.When the target is added,the binding of the target to the hairpin probe causes the neck ring structure to open and initiate the subsequent chain reaction.Subsequently,the two hairpin probes self-assemble into a highly ordered DNA double helix structure and are modified in the DNA.The fluorescent signal molecules on the double helix are also precisely synchronized to achieve the target's ultra-sensitive quantitative detection.Under the optimal conditions,there is a good linear relationship between the fluorescence and the concentration of MTB 16 sDNA ranging from 0.01 to 100 nM with the correlation coefficient of 0.993(n = 8)and the detection of limit 5.3 pM(3?/K).The base mispairing was also analyzed with the new method,and the results showed that present HCR method has the potential for single nucleotide polymorphism(SNP)analysis.Moreover,MBs-H1 can be reused simply without loss of sensitivity and is favor for reducing the test cost,which is profound important for MTB detection in poverty countries.Comparing with the present rapid methods based NAAT,the two sensitive and selective NAAT methods developed in this context are simple and rapid,and don't require complicated enzyme amplification and non-experts can also finish the detection.Which is important for MTB detection in poverty countries.