Comparison of P1 and 16SrRNA genes for Comparison of P1 and 16SrRNA genes for detection of Mycoplasma pneumoniae detection of Mycoplasma pneumoniae by optimized nested PCR in adult by optimized nested PCR in adult patients in Zhejiang, China patients in Zhejiang, China汇报人：周子博汇报人：周子博lMycoplasma pneumoniae is a frequent cause of community-acquired pneumonia for 10-40% in children and adults.lBecause of the treatment of M. pneumonia infection 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. lConventional tests for detecting M. pneumoniae have their limitations.IntroductionIntroductionlSeveral PCR-related methods provide enhanced sensitivity and have been successfully applied for research purposes such as nested PCR.lThe P1 adhesion gene and the 16SrRNA gene have been utilized widely in PCR techniques as the targets for detection of M. pneumoniae.lIn 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.MaterialsandmethodslStrains and clinical sampleslDNA preparationlOrthogonal array designlOptimization of single factor conditionslNested PCR sensitivity testlDetection of clinical samplesOrthogonal array designFactorsLevels123Primer() 0.1 0.3 0.5Mg2+(mM) 1.5 2.5 4Annealingtemperature() 58(54) 60(56) 62(58)Dilutionmultiple NO 50 100Table 1 Nested PCR factors and their levels for orthogonal projects (The annealing temperature of 16SrRNA gene is expressed in the brackets)Orthogonal array designFactorsReactionPrimer()Mg2+(mM)AnnealingDilutionmultipletemperature()10.11.558(54)NO20.12.560(56)5030.1462(58)10040.31.560(56)10050.32.562(58)NO60.3458(54)5070.51.562(58)5080.52.558(54)10090.5460(56)NOTable 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 pneumoniae FH with the target of the P1 adhesion (16SrRNA )gene. M 1 2 3 4 5 6 7 8 9100bp150bp107bpM 1 2 3 4 5 6 7 8 9144bp150bpP1gene16SrRNASinglefactorexperiment 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 dilution; 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.P1gene16SrRNANestedPCRsensitivitytestsensitivities 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: 10-2 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 P1gene:1pg 1 2 3 4 5 6 7 8 912345678916SrRNA；DetectionofclinicalsamplesP1 adhesion gene:( 25/55; 43.6%)Detectionofclinicalsamples16SrRNA gene (30/55 ;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 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 conditions 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 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 16SrRNA gene is the most excellent target for detection of M. pneumoniae by nested PCR.discussionIn this study, we adopted nested PCR to compare the two targets. The superior sensitivity is the major advantage of nested PCR. 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 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 than that for single-step PCR and exceeds that for antigen capture enzyme immunoassay and culture by 104- to 105-fold.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 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 usingthe standard strain of M. pneumoniae, since orthogonal test design can greatly shorten the test number and can quickly arrive at a more appropriate reaction condition. Then we also utilizeda completely single factor test design based on the results of the orthogonal design. At last, the final optimal reaction conditions 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 laboratories.discussionIn 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 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 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 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 weakened 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 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.discussionThe sensitivity of nested 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 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 inspection 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. pneumoniae. 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 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 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.discussionAccording 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), for both P1 adhesion 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 patients 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 16SrRNA 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 16SrRNA ones.