肺炎支原體巢式PCR的優(yōu)化課件

1、Comparison of P1 and 16SrRNA genes for detection of Mycoplasma pneumoniae by optimized nested PCR in adult patients in Zhejiang, China匯報(bào)人：周子博Mycoplasma pneumoniae is a frequent cause of community-acquired pneumonia for 10-40% in children and adults.Because of the treatment of M. pneumonia infection

2、with -lactam antibiotics is ineffective and the clinical manifestations of M. pneumoniae infection are complicated and nonspecific, so a rapid, sensitive and specific laboratory test is vital for early diagnosis of M. pneumoniae infection. Conventional tests for detecting M. pneumoniae have their li

3、mitations.IntroductionIntroductionSeveral PCR-related methods provide enhanced sensitivity and have been successfully applied for research purposes such as nested PCR.The P1 adhesion gene and the 16SrRNA gene have been utilized widely in PCR techniques as the targets for detection of M. pneumoniae.I

4、n this study, we sought to identify the more sensitive and specific target (P1 or 16SrRNA) in M. pneumoniae detection and to evaluate the use of nested PCR for the diagnosis of MP infection from patients in whom M. pneumoniae was suspected.Materials and methodsStrains and clinical samplesDNA prepara

5、tionOrthogonal array designOptimization of single factor conditionsNested PCR sensitivity testDetection of clinical samplesOrthogonal array designFactors Levels 1 2 3Primer () 0.1 0.3 0.5Mg2+ (mM) 1.5 2.5 4Annealing temperature() 58(54) 60(56) 62(58)Dilution multiple NO 50 100Table 1 Nested PCR fact

6、ors and their levels for orthogonal projects (The annealing temperature of 16SrRNA gene is expressed in the brackets)Figure 1: Electrophoresis analysis of varied nested PCR products of Mycoplasma pneumoniae FH with the target of the P1 adhesion (16SrRNA )gene. M 1 2 3 4 5 6 7 8 9100bp150bp107bpM 1 2

7、 3 4 5 6 7 8 9144bp150bpP1 gene16SrRNASingle factor experiment At last, we determined the final optimal nested PCR reaction conditions: For the P1 gene, the optimal combination of Mg2+ concentration 3mM, primer concentration 0.3uM, annealing temperature 60 , the first round PCR product 50-fold dilut

8、ion; For the 16S gene, the most excellent combination of Mg2+ concentration 3mM, primer concentration 0.3uM, annealing temperature 56 , the first round PCR product 50-fold dilution.P1 gene16SrRNADetection of clinical samplesP1 adhesion gene:( 25/55; 43.6%)Detection of clinical samples16SrRNA gene (3

9、0/55 ;56.3%)discussionWith the development of molecular biology techniques, PCR technology has become the most valuable method for rapid diagnosis of Mycoplasma pneumoniae infection. Both P1adhesion gene and 16SrRNA gene are widely used as targets for detection of M. pneumoniae by PCR. However, it i

10、s still inconclusive for which target is better and our effort is to find the most suitable one. In this study, we jointed orthogonal experiment and single factor tests to optimize several crucial conditions of nested PCR and finally concluded the optimum reaction conditions of the two targets. Then

11、 we detected the sensitivity of the two targets on the basis of the optimal conditions and the results showed that the 16SrRNA gene is more sensitive than the P1 adhesion gene. We also presented a study by using clinical specimens from adult patients and found that 16SrRNA gene has a higher positive

12、 rate than the P1 adhesion gene. So the 16SrRNA gene is the most excellent target for detection of M. pneumoniae by nested PCR.discussionAlthough nested PCR is a rapid and sensitive method for early diagnosis of M. pneumoniae infection, the impact of nested PCR reaction conditions is numerous and it

13、 is time-consuming to find the optimal condition. What is more, only on the basis of the optimum conditions can obtain a more accurate and objective results. So we adopted the orthogonal array design to optimize several crucial factors affecting the nested PCR using the standard strain of M. pneumon

14、iae, since orthogonal test design can greatly shorten the test number and can quickly arrive at a more appropriate reaction condition. Then we also utilized a completely single factor test design based on the results of the orthogonal design. At last, the final optimal reaction conditions of nested

15、PCR are determined by integrated the results of the methods above, so the comparison of the P1 adhesion gene and the 16SrRNA gene primers under this optimal condition can be more objective than that of other laboratories.discussionIn our study, Orthogonal array design was adopted to optimize four co

16、mmon factors affecting the nested PCR, which were the concentration of primers and Mg2+, dilution multiple of the first round PCR product, annealing temperature. All of them are the critical element in the performance of nested PCR. Firstly, through the test we found that excessively low primer conc

17、entration can reduce PCR yield and excessively high primer concentration increase the probability of mispriming and generations of non-specific PCR products. Secondly, form our experiments, If Mg2+ concentration was too low, the yield of PCR product could be reduced, since Tap DNA polymerases are Mg

18、2+-dependent enzyme and it is sensitive to the concentration of Mg2+. Thirdly, our results shows that poor specificity of amplified band appears at low annealing temperature, and weakened amplified bands at high annealing temperature. For the specificity of PCR mainly depends on annealing temperatur

19、e and improve the annealing temperature within a certain range can increase the specificity of the PCR reaction. Lastly, we also found that a lot of non-specific bands appear if not dilution, that was probably because the template concentration of second round of PCR is excessively high.discussionAc

20、cording to our results from clinical test, 16SrRNA gene proved clearly to be the best target for this purpose, yielding a positive PCR result in 56.3% of cases, while the positive rate was 43.6% for the P1 adhesion gene. The results also showed that there was an excellent correspondence of positive subjects detected by the P1 adhesion gene and 16SrRNA gene primers, and the coincidence rate of the two gene primers can reach 76.4%(42/55), fo