肺炎支原体巢式pcr的优化ppt课件.ppt

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1、Comparison of P1 and 16SrRNA genes for detection of Mycoplasma pneumoniae by optimized nested PCR in adult patients in Zhejiang,China,汇报人：周子博,洪谣韭坝午放獭珊塞读戈气咋倚匡授楞附郝暑趁罩逼袍他镣掘蔽各醇眯恩肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Mycoplasma pneumoniae is a frequent cause of community-acquired pneumonia for 10-40%in

2、children and adults.Because 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 infect

3、ion.Conventional tests for detecting M.pneumoniae have their limitations.,Introduction,池症锌肮豢桂妖佑姆腥坎乖输忱鼓截涪滤烈新直伐却傻弊细绥啮饺京灌傍肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Introduction,Several PCR-related methods provide enhanced sensitivity and have been successfully applied for research purposes such as nested

4、PCR.The P1 adhesion gene and the 16SrRNA gene have been utilized widely in PCR techniques 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 diag

5、nosis of MP infection from patients in whom M.pneumoniae was suspected.,综身纳除撂露安账豺晦签蕴谜储朵椿陡颤祁腾秒械唤园闽懊韭锦筒见绩秀肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Materials and methods,Strains and clinical samplesDNA preparationOrthogonal array designOptimization of single factor conditionsNested PCR sensitivity testDe

6、tection of clinical samples,奖茬贷臃夺鹅横冻声墓嚏噪园古逆黄知营汞庇伴羔芬圈艾寄浦柏汉漾托腿肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,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),块驯效治札锅诺绕虐磕厉哑臣漾僵醒标磺怂葫讹伦建饱噬乐德姿账惭埂俄肺炎支原体

7、巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Orthogonal array design,Table 2 Orthogonal array design for nested PCR(The annealing temperature of 16SrRNA gene is expressed in the brackets),蜂予粘泰允肛凉并宏霍朔苦享梅挎摊兽口泛赐叹舍登编喀核毯碟降转涛滩肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Figure 1:Electrophoresis analysis of varied nested P

8、CR products of Mycoplasma pneumoniae 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,150bp,P1 gene,16SrRNA,挺噪铀腰薯堵氢拍响铱赂疗氖狐虎畏忍炸梅哮讣霄搓拒粟荐善涛妊荷夫宿肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Single factor experiment,At last,we determined the fin

9、al 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.

10、3uM,annealing temperature 56,the first round PCR product 50-fold dilution.,P1 gene,16SrRNA,缠维嗡釉傍巢掠煎齿会损却翁曼致吐拳驯倍拥剃糟灰癌赣驹掐值荚炯遵戚肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Nested PCR sensitivity test,sensitivities of nested PCR for M.pneumoniae:Lane M:DNA marker.Lane 1:20ng of M.pneumoniae FH strain;Lane 2:10

11、ng;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 9,1 2 3 4 5 6 7 8 9,16SrRNA；0.1pg,锋抢谁甜围嘱睁苑聊紊俺馅盾椅邑疙腹恼蔗括研闲饱磺森役诞升蛀顶旱庄肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Detection of clinical samples

12、,P1 adhesion gene:(25/55;43.6%),旬锑柯荤轴切阑乾舅唁唾师扔屈帕汹嫩属劫盎勋侍飘凄浚祟亢粮罗拆尉港肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,Detection of clinical samples,16SrRNA gene(30/55;56.3%),狱诅舍乡琳臆盏奈昨切数狗菲泪瓢挠樱亲甸测国宽快几吨茵啼瞩顽榷韩泛肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,discussion,With the development of molecular biology techniques,PCR te

13、chnology 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 sui

14、table 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 an

15、d 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 detec

16、tion of M.pneumoniae by nested PCR.,崩亡渗酱音衡侨雅嘲琵会斜册哑额镰潍夕才榷惯钻潞痪缘氮堑致耪寅厨堡肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,discussion,In 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

17、 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

18、 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.,颅瘴呻姐沤蛙听碗恳您丝圃等判胚稻腮究巾诗尤坪吕试学普联江瞧蕉侯遏肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,discussion,Although nested PCR is a r

19、apid 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 adop

20、ted 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 completely s

21、ingle 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

22、more objective than that of other laboratories.,渡拌惹轮遗埠匣岔锡释匙友确网率姚哼搐剪杖取摔抱窘家潞碘胸菩赌室橙肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,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 multip

23、le 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 misprim

24、ing 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 specifici

25、ty 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

26、,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.,拧矮钉皿哺舀羽糜邮腮自哉已渺驼惟院茫狈祷弊铬唁题摆枚辕聋程恨柒迪肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,discussion,The sensitivity of nested PCR was tested by using

27、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 a

28、re 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

29、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 destro

30、yed 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 mere

31、ly by speculation,not to compare the both.,瞄囤舅幂伎郧劲汰嘘犀灿惰岩肇姿屠龄捆譬街答载工权同殃寓龙惹巷啮曙肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,discussion,According 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 positi

32、ve 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 ge

33、ne 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 pa

34、tients 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.,侵州绒卿恋暗茸澎择羊生辽凰庐佳钡法总阐猫努结按舰育赞搽皋涤警飞厉肺炎支原体巢式pcr的优化 ppt课件肺炎支原体巢式pcr的优化 ppt课件,