Development And Application Of Bacterial RT-qPCR Detection Technology Based On High Abundant Transcripts

In recent years, with the recurrence of tuberculosis and the emergence of super bacteria, it always remind people that infectious disease is still a major threat to human health. With the developing of science and technology, we have developed a variety of methods to detect and diagnose diseases, such as the traditional method of microbiological culture, immune detection, molecular biological detection methods, etc. Molecular biological detection method is becoming the core of modern detection especially the PCR(Polymerase Chain Reaction) as it is rapid, sensitive, specific and it also has a wide range of deformation. However, most of the deformation detect the genomic DNA as the detection target. These methods in the detection of the virus pathogenic microorganisms are very sensitive, but in the detection of bacterial pathogens have been limited. Because the copy number of the bacterial genome is often equal to the number of bacteria, the number of bacteria in the clinic is limited by the number of bacteria in the host, especially the intracellular parasitic bacteria, which limits the detection.With the expressing of the coding gene, the different abundance of the RNA will be transcribed. For the genes,the higher expression, the more abundance of m RNA. Therefore, if choose the had expressed genes and its m RNA as the detection target, it will greatly improve the sensitivity of detection for bacteria. According to the principle of the reverse transcription PCR, it is possible to meet the requirement that amplify the DNA and RNA at the same time.As a method to detect RNA, the Reverse transcription PCR has been widely used in the detection of viral microorganisms and alive bacteria. While the method has not yet used in the detection of pathogenic bacteria.RNA is extracted from Malta B.melitensis 16 M which is as a model in this experiment and sequence for the high expression genes. The principle of this approach is that assay sensitivity can be greatly improved by choosing highly transcribed genes as signature sequences and detecting both DNA and its RNA transcripts.In order to test the feasibility of RT-q PCR, we used the equal volume of 16 M bacteria to extract the equal volume of DNA and RNA. Comparing the amplification efficiency of DNA and RNA by RT-q PCR and q PCR, we confirmed that RT-q PCR efficiently amplifies both DNA and RNA, while q PCR amplifies only DNA. However, when comparing the amplification efficiency of RNA and DNA by RT-q PCR, we found that the CT value of RNA detected by RT-q PCR was much larger than DNA. This is not in conformity with the theory. Therefore, we compared the concentration changes of RNA before and after treatment with DNase I. The results showed that the RNA extracted by Trziol lysis method has a serious pollution by DNA and the concentration of the method is too low to test the RT-q PCR. Only 22.7% RNA is the pure molecule.In order to improve the detection efficiency of the RT-q PCR, we used a simple method of thermal denaturation to treat samples. Then using RT-q PCR and q PCR to detect the samples. Results showed that the CT value of RT-q PCR is lower than q PCR, which shows that the thermal denaturation method can avoid the loss of RNA.In order to further compare the detection sensitivity of RT-q PCR and q PCR using heat treated, the samples diluted 10 times and detected by RT-q PCR and q PCR. Results show that all the RT-q PCR are better than q PCR.In order to find the appropriate target genes for detection of 16 M, we analyzed other candidate genes. The results showed that the gene BMEI1305 is the best and can be used RT-q PCR for the detection of 16 M. To evaluate the sensitivity of RT-PCR for clinical sample detection, blood samples were collected from brucellosis patients and detected using q PCR and RT-q PCR assays. In the 10 samples, four samples were found to be positive using q PCR, while six were found to be positive using RT-q PCR. These data indicate that in the diagnosis of brucellosis, RT-q PCR is better than q PCR detection. To test whether this technique can be applied universally for bacterial detection, five other bacteria were also detected. Candidate target genes with high transcription levels were selected based on previously reported expression profile studies. Bacterial cultures were heat lysed and detected using q PCR and RT-q PCR. For the selected genes, the RT-q PCR Ct values were lower than those of q PCR. These data confirmed that RT-q PCR for sensitive detection of bacterial pathogens could be extended to other bacteria and be used as a universal strategy for bacterial detection.In conclusion, the RT-q PCR based on the transcripts has the advantages of high sensitivity and specificity and it can be used in clinical diagnosis.