肺炎支原體巢式pcr的優(yōu)化

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 communi

2、ty-acquired pneumonia for 10-40% in children and adults.Because of the treatment of M. pneumonia infection with β-lactam antibiotics is ineffective and the clinical manifestations of M. pneumoniae infection are complica

3、ted 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 limitations.,Introduction,Introd

4、uction,Several 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 tec

5、hniques as the targets for detection of M. pneumoniae.In 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

6、 diagnosis of MP infection from patients in whom M. pneumoniae was suspected.,Materials and methods,Strains and clinical samplesDNA preparationOrthogonal array designOptimization of single factor conditionsNested PCR

7、 sensitivity testDetection of clinical samples,Orthogonal array design,Table 1 Nested PCR factors and their levels for orthogonal projects (The annealing temperature of 16SrRNA gene is expressed in the brackets),Orthog

8、onal array design,Table 2 Orthogonal array design for nested PCR (The annealing temperature of 16SrRNA gene is expressed in the brackets),,Figure 1: Electrophoresis analysis of varied nested PCR products of Mycoplasma p

9、neumoniae FH with the target of the P1 adhesion (16SrRNA )gene.,M 1 2 3 4 5 6 7 8 9,100bp→,150bp→,←107bp,M 1 2 3 4 5 6 7 8 9,144bp,150

10、bp→,P1 gene,16SrRNA,Single 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, anneal

11、ing temperature 60 ℃, the first round PCR product 50-fold dilution; For the 16S gene, the most excellent combination of Mg2+ concentration 3mM, primer concentration 0.3uM, annealing temperature 56 ℃, the first round PCR

12、product 50-fold dilution.,P1 gene,16SrRNA,Nested PCR sensitivity test,sensitivities of nested PCR for M. pneumoniae: Lane M: DNA marker. Lane 1: 20ng of M. pneumoniae FH strain; Lane 2: 10ng; Lane 3: 10-1 ng; Lane 4: 10-

13、2 ng; Lane 5: 10-3 ng; Lane 6: 10-4 ng; Lane 7: 10-5 ng; Lane 8: 10-6 ng; Lane 9: negative control.,M 1 2 3 4 5 6 7 8 9,P1 gene: 1pg,1 2 3 4 5 6 7 8 9,1 2 3

14、 4 5 6 7 8 9,16SrRNA； 0.1pg,Detection of clinical samples,P1 adhesion gene:( 25/55; 43.6%),Detection of clinical samples,16SrRNA gene (30/55 ;56.3%),discussion,With the development of molecular biology te

15、chniques, 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.

16、 However, it is 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 condition

17、s of nested PCR and finally concluded the optimum reaction conditions of the two targets. Then we detected the sensitivity of the two targets on the basis of the optimal conditions and the results showed that the 16SrRNA

18、 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 rate than the P1 adhesion gene. So the 16SrRN

19、A gene is the most excellent target for detection of M. pneumoniae by nested PCR.,discussion,In this study, we adopted nested PCR to compare the two targets. The superior sensitivity is the major advantage of nested PCR.

20、 The sensitivity can be increased by nested PCR because it involves the reamplification of a PCR product with a second primer set. Nested PCR may lead to a 103-fold increase in sensitivity than single-step PCR, as nested

21、 PCR enables the detection of 1-100fg of DNA, and single-step PCR assays can only detect 10-100pg of DNA. Abele-Horn et al. can detect 30-100fg of M. pneumoniae DNA by nested PCR, and the sensitivity is 103-fold better t

22、han that for single-step PCR and exceeds that for antigen capture enzyme immunoassay and culture by 104- to 105-fold.,discussion,Although nested PCR is a rapid and sensitive method for early diagnosis of M. pneumoniae in

23、fection, the impact of nested PCR reaction conditions is numerous and it 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

24、 results. So we adopted the orthogonal array design to optimize several crucial factors affecting the nested PCR using the standard strain of M. pneumoniae, since orthogonal test design can greatly shorten the test numb

25、er 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 conditio

26、ns of nested 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

27、 laboratories.,discussion,In our study, Orthogonal array design was adopted to optimize four common factors affecting the nested PCR, which were the concentration of primers and Mg2+, dilution multiple of the first round

28、 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 concentration can reduce PCR yield and excessivel

29、y 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 reduce

30、d, since Tap DNA polymerases are Mg2+-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 weaken

31、ed amplified bands at high annealing temperature. For the specificity of PCR mainly depends on annealing temperature and improve the annealing temperature within a certain range can increase the specificity of the PCR re

32、action. 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.,discussion,The sensitivity of nested

33、 PCR was tested by using serial dilutions (1:10) of M. pneumoniae DNA, our results suggested that the 16SrRNA gene primers were more sensitive than the P1 adhesion gene primers, as the 16SrRNA gene primers can detect up

34、to 0.1pg of M. pneumoniae DNA and the P1 gene primers can detect 1pg of M. pneumoniae DNA at most. Our findings are well confirmed to the study by Mohamed Nour et al, who found that the fragment intensity after visual in

35、spection of gels was always higher with 16SrDNA primers than with those directed to P1 adhesion gene, this showed that the amplification of the 16SrRNA gene by nested PCR were more sensitive for the detection of M. pneum

36、oniae. This was mainly because the presence of approximately 103 copies of 16SrRNA per mycoplasma cell and the high degree of conservation of the rRNA genes allowing a highly fixation of primers on the target and lead to

37、 a higher PCR yield. What is more, due to the RNA is destroyed more rapidly than the DNA after the death of the mycoplasma cell, detection of RNA provides further evidence of viable mycoplasmas in the specimen. K. Loens

38、et al. thought that the P1adhesion gene primers were found to be more sensitive than the 16SrRNA ones. However, they come to this conclusion merely by speculation, not to compare the both.,discussion,According to our res

39、ults 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

40、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), for both P1 adhes

41、ion gene and 16SrRNA gene were the ideal targets for PCR as both of them were the highly conservative repetitive sequence. The major difficulties for the interpretation of the PCR date were the discordant result, nine pa

42、tients were positive by the 16SrRNA gene primers but negative by the P1 adhesion gene primers and four patients were positive by the P1 adhesion gene but negative by the 16SrRNA ones. The former mainly because the 16SrRN

43、A gene primers are more sensitive than the P1 adhesion gene, as it can be proved by the sensitive test we accomplished before. The latter possibly due to the P1 adhesion gene primers are less specific than that of 16SrRN