Detection Of The MRNA Of Mycobacterium Tuberculosis With Reverse Transcription PCR And Its Primary Application In Rifampin Rapid Susceptibility Test

Research backgrounds:The resurgence of tuberculosis has attracted worldwide attention to the need for more methods of rapid diagnosis and susceptibility test. Tuberculosis has become to be the leading killer among infectious diseases due to the outbreaks of multi-drug resistant tuberculosis (MDR-TB) and the epidemic together with human immunodeficiency virus (HIV). At present, to perform directly observed treatment (DOT) project immediately and reasonably is the key point in the control of tuberculosis. It requires the clinical microbiology laboratories to provide the diagnosis and drug susceptibility data of Mycobacterium tuberculosis as soon as possible. In America, the Centers for Disease Control and Prevention recommend that all isolates of Mycobacterium tuberculosis be tested for their susceptibility to antibiotics, and that susceptibility data for first-line drugs be available within 30 days after receipt of a specimen. But conventional culture-based techniques for diagnosis and susceptibility all have the shortcoming of a long turnround time. Although both radiometric and non-radiometric liquid culture systems have significantly shortened the turnround period, the results are still not available for 5-12 days after receipt of an isolate. In recent years, with the development of molecular biological techniques, gene diagnosis techniques, especially PCR, have become popular for their rapidness, sensitivity and specificity virtues. However, severalproblems must be resolved before PCR can be widely accepted in clinical application to patients with tuberculosis, such as the risk of obtaining false-positive results due to contamination of clinical specimens with M tuberculosis DNA product from the PCR laboratory, the inability of the PCR method to detect a difference between viable and ncnviable organisms, and the incapacity of the PCR method to determine drug susceptibility. Considerable work has already gone into resolving the problem of contamination. So many people began to focus their attention on finding good molecule markers for assessing microbial viability, such as ATP, 16s RNA and mRNA. The mRNA molecule is considered to be the promising one for its short half-life and ability to distinguish viable and dead bacterium. Hellyer et al. has studied the changes of mRNA, rRNA and DNA during anti-tuberculosis therapy and drawn a conclusion that mRNA should be a good molecule marker for viable Mycobacterium tuberculosis diagnosis compared with DNA and rRNA. Based on this theory, we have developed a rapid and sensitive method to detect the mRNA of the M. tuberculosis and investigated its application in rifampin rapid susceptibility. Objectives:To develop a rapid and sensitive detection method of Mycobacterium tuberculosis mRNA; study all the parameters of rifampin rapid susceptibility test through detection of Mycobacterium tuberculosis mRNA and draw a protocol of rifampin new molecular biological susceptibility test method. Compare the results of 20 clinical isolates of rifampin susceptibility by the method we set up and Bact/ALERT 3D auto culture system. Evaluate the value of detection Mycobacterium tuberculosis mRNA in rifampin rapid susceptibility. Materials and methods: 1. The first part1.1 To develop a method to detect M. tuberculosis a antigen 85Bmessenger RNA with the RT-PCR technique.M tuberculosis H37Rv (ATCC 27294) was selected as standard strain. Bacterium suspension was obtained when the strains of M. tuberculosis grew to the mid-logarithmic phase in Middlebrook 7H9 broth for 7 to 14 days; Total RNA of M tuberculosis was isolated with Tripure total RNA isolation reagents associated with ultrasonic disruption; The genomic DNA contamination in total RNA was digested with RNA free DNase; A pair of primers was designed according to the specific sequence encoding alpha antigen 85B. 130 bp product was obtained through amplification of a antigen 85B messenger RNA using reverse transcription PCR and gel electrophoresis. 1.2 To assess the sensitivi...