Development Of The Real-time Fluorescent Loop-mediated Isothermal Amplification(LAMP) And Reverse Dot Blot Hybridization For Mycobacterium Tuberculosis

Tuberculosis is a serious and harm infectious disease to human health. It is one ofthe major diseases focused by our disease control department as well as a global socialproblem concerned in public health. According to the world health organization (WHO),there are about860million people worldwide infected by Mycobacterium tuberculosis,1.3million of them died of tuberculosis.The incidence of tuberculosis is growing at anaverage annual increase rate of0.4%. Thus, it is essential to look for a fast and accuratemethod for the detection of Mycobacterium tuberculosis. Loop-mediated isothermalamplification(LAMP) is a rapid technique with high sensitivity and specificity. Reversedot blot(RDB) hybridization technique combines gene amplification and hybridizationtechnique, it has the advantagement of simple operation and appears accurate visualobservation. This study has established RDB method and LAMP method for detectionof Mycobacterium tuberculosis. The method supposes to meet the need of on-site rapiddetection and provide technical support for the prevention and control of tuberculosis.The study consists of four parts:Part Ⅰ: Cloning and sequence analysis of Mycobacterium tuberculosis insertionsequence IS6110 A specific Mycobacterium tuberculosis inserted sequence IS6110has been chosenas the target sequence, to design a pair of specific primers from IS6110gene sequencein GenBank(Genbank NO:X17348.1). The amplified fragment through PCR wasinserted into the vector pGEM-T. The cloned plasmid pGEM-T-IS6110was obtainedafter transformed into E.coli competent cells and blue-white spot screening. It wasdemonstrated that the cloned plasmid pGEM-T-IS6110from Mycobacteriumtuberculosis has been successfully inserted to the sequence IS6110after theidentification by PCR and sequencing.Part Ⅱ: Development of real-time fluorescent LAMP detection method forMycobacterium tuberculosisThe LAMP primer group (including a pair of inner primers and outer primers) wasdesigned and synthesized by Mycobacterium tuberculosis IS6110gene. All primerswere mixed in a certain proportion of solution concentration to establish real-timefluorescent LAMP detection method for Mycobacterium tuberculosis. The molarconcentration ratio of inner and outer primers, as well as reaction temperature wereoptimized in order to obtain the optimum reaction conditions. The results were analyzedby the amplification curves. The results indicated that the whole detection wascompleted in1h and most reactions was amplified within30min. Real-time fluorescentLAMP detection method was proved to be sensitive and specific. The result was notinterfered by other common pathogens. The detection limit was2.4fg/reaction which is100-fold higher than PCR. So, this method has a wide prospect in clinical application.Part Ⅲ: Development of reverse dot blot hybridization assay for MycobacteriumtuberculosisSpecific oligonucleotide probe and biotinylated primers were designed andsynthesized according to Mycobacterium tuberculosis IS6110gene. The amplifiedbiotinylated fragment was obtained after PCR. The biotinylated PCR product wasdenatured and then hybridized with oligonucleotide probe on the positively chargednylon membrane at65℃.Each hybridization was processed with a positive control anda negative control. The probe concentration, streptavidin alkaline phosphatase dilution, hybridization time, enzymatic reaction time were optimized. The results were analysisedby the presence of visible purple-blue spots on the membrane after color reaction. Thereverse dot blot hybridization assay has highly specificity and no cross-reacrion withother common pathogens.24fg of Mycobacterium tuberculosis DNA in each reactioncan be detected with10-fold higher than PCR. Moreover, this method can realize thedifferential diagnosis between Mycobacterium tuberculosis and other commonpathogens.Part Ⅳ: The application of real-time fluorescent LAMP and reverse dot blothybridization in clinical specimensThe sputum samples DNA from clinically diagnosed tuberculosis patients weredetected by real-time fluorescent LAMP and reverse dot blot hybridization. All sputumsamples had fluorescent amplification curves and purple-blue spots. It has indicated thatthis two methods detect Mycobacterium tuberculosis successfully. The method has beenproved as a sensitive and specific method without using precise equipment and shouldbe promoted in clinical laboratories.