1、学习学习肺炎支原体巢式肺炎支原体巢式pcr的的优化优化l Mycoplasma pneumoniae is a frequent cause of community-acquired pneumonia for 10-40%in children and adults.l Because of the treatment of M.pneumonia infection with-lactam antibiotics is ineffective and the clinical manifestations of M.pneumoniae infection are complicated
2、 and nonspecific,so a rapid,sensitive and specific laboratory test is vital for early diagnosis of M.pneumoniae infection.l Conventional tests for detecting M.pneumoniae have their limitations.IntroductionIntroductionIntroductionl Several PCR-related methods provide enhanced sensitivity and have bee
3、n successfully applied for research purposes such as nested PCR.l The P1 adhesion gene and the 16SrRNA gene have been utilized widely in PCR techniques as the targets for detection of M.pneumoniae.l In this study,we sought to identify the more sensitive and specific target(P1 or 16SrRNA)in M.pneumon
4、iae 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 methodsMaterials and methodsl Strains and clinical samplesl DNA preparationl Orthogonal array designl Optimization of single factor conditionsl Nested
5、PCR sensitivity testl Detection 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 gen
6、e 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 PC
7、R(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 9144bp150bpP1 gene16SrRNASingle fac
8、tor experimentSingle 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 dilution;For the 16S gene,the most ex
9、cellent combination of Mg2+concentration 3mM,primer concentration 0.3uM,annealing temperature 56,the first round PCR product 50-fold dilution.P1 gene16SrRNANested PCR sensitivity testNested PCR sensitivity testsensitivities of nested PCR for M.pneumoniae:Lane M:DNA marker.Lane 1:20ng of M.pneumoniae
10、 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 P1 gene:1pg 1 2 3 4 5 6 7 8 91 2 3 4 5 6 7 8 916SrRNA；0.1pgDetection of clinical Detection of clinical samplessamples P1 adhesion gene:(25/55;4
11、3.6%)Detection of clinical Detection of clinical samplessamples 16SrRNA 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
12、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 fina
13、lly 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 f
14、rom 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 ma
15、jor 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
16、-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 neste
17、d 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 result
18、s.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 number and can quickly arrive at a more appropriate reaction condition.Then we also utilized a
19、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 condi
20、tion can be more objective than that of other 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 PCR product,annealing temperature.All
21、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,fo
22、rm 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,an
23、d 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 no
24、t 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 adhesio
25、n 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
26、 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
27、 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.
