围产期感染沙眼衣原体MOMP基因裂解酶片断长度多态性研究
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摘要
目的 探讨沙眼衣原体（Ct）套式聚合酶链反应（nPCR）、DNA纯化、测序的检测方法；首次建立Ct上游引物5′末端地高辛(Dig)标记裂解酶片段长度多态性（CFLP）分型方法并探索和优化Ct主要外膜蛋白（MOMP）基因（ompl）第Ⅳ可变区（VDⅣ）变温CFLP条件;了解重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型。
方法 取重庆市妇幼保健院2003年4月~2004年2月临产孕妇宫颈刮片及其新生儿鼻咽部拭子组成母婴配对标本300对共605份，采用Ct ompl套式聚合酶链反应（ompl-nPCR）和用内套引物直接扩增（ompl–PCR）及共有质粒（plasmid）-PCR检测Ct，用扩大金标准判断结果；用透析袋电泳洗脱法纯化DNA；用ABI PRISM 377型全自动DNA 测序仪测序；首次建立上游引物5′末端Dig标记CFLP方法并进行临床Ct分离株的基因分型。
结果 ompl-nPCR和ompl-PCR最低检测限分别为2.5EB和25EB，即前者比后者灵敏度高10倍。其敏感性分别为100%（33/33）和87.9%
（29/33），特异性分别为99.6%（266/267）、99.3%（265/267）。孕妇宫颈Ct检出率为11%（33/300），其所生的305例新生儿Ct阳性8例，母婴传播率24.2%（8/33）。生后24h内和5~10d采集宫颈Ct阳性母亲所生新生儿鼻咽部拭子标本各33份和18份，检出Ct阳性1例和7例,检出率分别为3.0%（1/33）和38.9%（7/18），χ2=5.73，P<0.025。33例Ct阳性孕妇，剖宫产24例，其子 Ct阳性2例，剖宫产母婴传播率8.3%（2/24）；阴道分娩9例，其子Ct阳性6例，阴道分娩母婴传播率66.7%（6/9），χ2=4.43，P<0.05。在Ct阳性和阴性孕妇中，胎膜早破发生率分别为30.3%（10/33）和13.5%（36/267），χ2=4.2，P<0.05。PCR产物经透析袋电泳洗脱法纯化后，电泳条带清晰、没有非特异显影，起始部没有杂质残留，19例临床标本用分光光度法测纯化DNA浓度为0.1μg/μl ~0.25μg/μl，全部达到测序和进行CFLP分析要求。8对Ct阳性母婴配对标本CFLP图谱呈四类，经测序证实分别为E、F、H、D型（各3、2、2、1对），分别占37.5%、25%、25%和 12.5%，且每对母子CFLP图谱完全一致；同一标本不同批次产生的CFLP图谱和相同基因型的不同标本的CFLP图谱相同，显示出良好的重复性；本实验显示Ct ompl VDⅣ CFLP条件以8%变性聚丙烯酰胺凝胶电泳、40℃退火温度、10min~15min酶切时间获得的电泳图谱效果较为理想。
结论 本实验结果在一定意义上反映了重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型；上游引物5′末端Dig标记Ct ompl VDⅣ CFLP基因分型方法具有灵敏度高、重复性好、操作
简便、快速、花费相对低廉，没有放射污染等特点，是一种极具潜力的Ct基因分型方法。
Objective To establish the methed of cleavase fragment length polymorphism (CFLP) analysis labeled at the 5′-end of one primer with digoxigenin for genotyping of chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein （MOMP）gene (ompl) nested polymerase chain reaction (ompl-nPCR), DNA purification and DNA sequencing were studied and compared. The incidence of Ct infection in pregnant women, the common genotypes and vertical transmission rate of Ct in Chongqing area during the past one year were also investigated.
Methods The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from Apr. 2003 to Feb. 2004 in Chongqing Women and Children’s Health Care Institute. Total 300 pairs (605 specimen) were detected by ompl-nPCR, ompl-PCR (inside pair
of primers was used directly) and plasmid-PCR. The results were judged by the modified gold standard (MGS). The ompl-nPCR DNA was purificated by recovery of DNA from agarose gels electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay labeled at the 5′-end of one primer with digoxigenin was created for genotyping of Ct, and was primarily applied.
Results The minimum detectable levels of ompl-nPCR and ompl-PCR were 2.5 EB and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times of ompl-PCR. The sensitivity rate of ompl-nPCR and ompl-PCR were 100%（33/33）and 87.9%（29/33）, respectively. The specificities were 99.6%（266/267）and 99.3%（265/267）, respectively. The positive rate of Ct in the samples from the pregnant women was 11% (33/300). The vertical transmission rate of Ct from mothers to their infants was 24.2% (8/33). The nasopharyngeal swabs taken in 24 hour and 5~10 days after birth were detected. The positive detected rate of Ct in the later samples was 38.9% (7/18), which was significantly greater than that of 3.0% (1/33) in the samples of first 24 hours (χ2=5.73，p<0.025). Of the 33 Ct-positive samples from pregnant women 9 had vaginal delivery and 24 had caesarean section. The vertical transmission rates in vaginal delivery group and caesarean section group were 66.7% (6/9) and 8.3% (2/24),
respectively. The rate of premature rupture of membrane in Ct-positive group was 30.3% (10/33), which was greater than that of Ct-negative groups (13.5%, 36/267, χ2=4.2，p<0.05).
Four kinds of different patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants by using CFLP, which were confirmed by DNA sequencing later. They are type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair). Each pair of matched maternal-infantile samples presented identical CFLP pattern.
Conclusions These study revealed the infection rate of Ct in pregnant women, vertical transmission rate of Ct and the common genotypes of Ct in Chongqing Women and Children’s Health Care Institute. The CFLP assay labeled at the 5′-end of one primer with digoxigenin was first used for genotyping of Ct. It showed the good sensitivity and reproducibility, no radioactive contamination, and is simple. It was a potential new method for Ct genotyping.
方法 取重庆市妇幼保健院2003年4月~2004年2月临产孕妇宫颈刮片及其新生儿鼻咽部拭子组成母婴配对标本300对共605份，采用Ct ompl套式聚合酶链反应（ompl-nPCR）和用内套引物直接扩增（ompl–PCR）及共有质粒（plasmid）-PCR检测Ct，用扩大金标准判断结果；用透析袋电泳洗脱法纯化DNA；用ABI PRISM 377型全自动DNA 测序仪测序；首次建立上游引物5′末端Dig标记CFLP方法并进行临床Ct分离株的基因分型。
结果 ompl-nPCR和ompl-PCR最低检测限分别为2.5EB和25EB，即前者比后者灵敏度高10倍。其敏感性分别为100%（33/33）和87.9%
（29/33），特异性分别为99.6%（266/267）、99.3%（265/267）。孕妇宫颈Ct检出率为11%（33/300），其所生的305例新生儿Ct阳性8例，母婴传播率24.2%（8/33）。生后24h内和5~10d采集宫颈Ct阳性母亲所生新生儿鼻咽部拭子标本各33份和18份，检出Ct阳性1例和7例,检出率分别为3.0%（1/33）和38.9%（7/18），χ2=5.73，P<0.025。33例Ct阳性孕妇，剖宫产24例，其子 Ct阳性2例，剖宫产母婴传播率8.3%（2/24）；阴道分娩9例，其子Ct阳性6例，阴道分娩母婴传播率66.7%（6/9），χ2=4.43，P<0.05。在Ct阳性和阴性孕妇中，胎膜早破发生率分别为30.3%（10/33）和13.5%（36/267），χ2=4.2，P<0.05。PCR产物经透析袋电泳洗脱法纯化后，电泳条带清晰、没有非特异显影，起始部没有杂质残留，19例临床标本用分光光度法测纯化DNA浓度为0.1μg/μl ~0.25μg/μl，全部达到测序和进行CFLP分析要求。8对Ct阳性母婴配对标本CFLP图谱呈四类，经测序证实分别为E、F、H、D型（各3、2、2、1对），分别占37.5%、25%、25%和 12.5%，且每对母子CFLP图谱完全一致；同一标本不同批次产生的CFLP图谱和相同基因型的不同标本的CFLP图谱相同，显示出良好的重复性；本实验显示Ct ompl VDⅣ CFLP条件以8%变性聚丙烯酰胺凝胶电泳、40℃退火温度、10min~15min酶切时间获得的电泳图谱效果较为理想。
结论 本实验结果在一定意义上反映了重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型；上游引物5′末端Dig标记Ct ompl VDⅣ CFLP基因分型方法具有灵敏度高、重复性好、操作
简便、快速、花费相对低廉，没有放射污染等特点，是一种极具潜力的Ct基因分型方法。
Objective To establish the methed of cleavase fragment length polymorphism (CFLP) analysis labeled at the 5′-end of one primer with digoxigenin for genotyping of chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein （MOMP）gene (ompl) nested polymerase chain reaction (ompl-nPCR), DNA purification and DNA sequencing were studied and compared. The incidence of Ct infection in pregnant women, the common genotypes and vertical transmission rate of Ct in Chongqing area during the past one year were also investigated.
Methods The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from Apr. 2003 to Feb. 2004 in Chongqing Women and Children’s Health Care Institute. Total 300 pairs (605 specimen) were detected by ompl-nPCR, ompl-PCR (inside pair
of primers was used directly) and plasmid-PCR. The results were judged by the modified gold standard (MGS). The ompl-nPCR DNA was purificated by recovery of DNA from agarose gels electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay labeled at the 5′-end of one primer with digoxigenin was created for genotyping of Ct, and was primarily applied.
Results The minimum detectable levels of ompl-nPCR and ompl-PCR were 2.5 EB and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times of ompl-PCR. The sensitivity rate of ompl-nPCR and ompl-PCR were 100%（33/33）and 87.9%（29/33）, respectively. The specificities were 99.6%（266/267）and 99.3%（265/267）, respectively. The positive rate of Ct in the samples from the pregnant women was 11% (33/300). The vertical transmission rate of Ct from mothers to their infants was 24.2% (8/33). The nasopharyngeal swabs taken in 24 hour and 5~10 days after birth were detected. The positive detected rate of Ct in the later samples was 38.9% (7/18), which was significantly greater than that of 3.0% (1/33) in the samples of first 24 hours (χ2=5.73，p<0.025). Of the 33 Ct-positive samples from pregnant women 9 had vaginal delivery and 24 had caesarean section. The vertical transmission rates in vaginal delivery group and caesarean section group were 66.7% (6/9) and 8.3% (2/24),
respectively. The rate of premature rupture of membrane in Ct-positive group was 30.3% (10/33), which was greater than that of Ct-negative groups (13.5%, 36/267, χ2=4.2，p<0.05).
Four kinds of different patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants by using CFLP, which were confirmed by DNA sequencing later. They are type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair). Each pair of matched maternal-infantile samples presented identical CFLP pattern.
Conclusions These study revealed the infection rate of Ct in pregnant women, vertical transmission rate of Ct and the common genotypes of Ct in Chongqing Women and Children’s Health Care Institute. The CFLP assay labeled at the 5′-end of one primer with digoxigenin was first used for genotyping of Ct. It showed the good sensitivity and reproducibility, no radioactive contamination, and is simple. It was a potential new method for Ct genotyping.
引文
Mardh PA. Influence of infection with Chlamydia trachomatis on pregnancy outcome, infant health and life-long sequelae in infected offspring. Best Pract Res Clin Obstet Gynaecol 2002; 16: 847-64.
Andrews WW, Goldenberg RL, Mercer B, et al. The Preterm Prediction Study: association of second-trimester genitourinary Chlamydia trachomatis infection with subsequent spontaneous prêt, rm birth. Am J Obstet Gynecol 2000; 183:662-668.
Poole E, Lamont. Chlamydia trachomatis serovar differentiations by direct sequence analysis of the variable segment 4 region of the major outer membrane protein. Infect Immune 1992; 60(3): 1089.
Schachter J, Stamm W, Chernesky MA, et al. Nonculture tests for genital tract Chlamydial infection. Sex Tran Dis 1992; 19:243-244.
Lebar WD. Keeping up with new technology: new approaches to diagnosis of Chlamydial infection. Clin Chem 1996,42(5): 809-812.
Kessler HH, Pierer K, Stuenzner D, et al. Rapid detection of Chlamydial trachomatis in conjunctival, pharyngeal, and urethral specimens with a new polymerase chain reaction assay. Sex Tran Dis 1994,21(4): 191-195.
俞信忠,许平，张国荣.绍兴地区临产前孕妇沙眼衣原体感染的调查.中国优生与遗传杂志,1999,7(1):30.
陈仕红,妊娠期妇女沙眼衣原体感染的检测报告.中国优生与遗传杂志,1999,7(4):28.
苏亦平,花玉波,金红,等.延边地区孕妇沙眼衣原体感染研究.中国实验诊断学,1998,2(3):121.
应长青,王滨有,郑冬梅,等.孕妇沙眼衣原体感染与妊娠结局及新生儿发病的关系.中华妇产科杂志,1999,34(6):384-350.
张健,卢静,孕妇沙眼衣原体感染与新生儿感染关系的研究.济宁医学院学
报,2002,25(1):55-56.
童皎,围产期沙眼衣原体感染研究.新生儿科杂志,2003,18(4):148-150.
黄艳丽,沙眼衣原体感染与妊娠结局的临床观察.中华医院感染学杂志,2001,11(2):111.
李玉梅,鲁继荣,傅文永,等. 沙眼衣原体垂直传播基因水平的快速检测.临床儿科杂志,2000,18(3):133-134.
鲁继荣, 李玉梅, 傅文永,等. 沙眼衣原体母婴垂直传播的研究.中华儿科杂志,1999,37(12):739-741.
张春平,朱道银,郭晓霞.沙眼衣原体子宫内感染途径的研究.中华妇产科杂志,2002,37(3):149-151.
吴仕孝,沈犁,刘官信.母婴垂直传播沙眼衣原体多聚酶链反应及测序研究.中华儿科杂志,1997,35(6):283-285.
Pao CC, Kao SM, Wang HC, et al. Intraumniotic of Chlamydia trachomatis deoxyribonucleic acid sequences by polymerase chain reaction. Am J Obstet Gynecol, 1991, 164: 1295-1299.
丁瑛,顾惠珍,龚增娥,等. 孕妇及新生儿沙眼衣原体的临床观察.中华妇产科杂志,1995,30(2):74-76.
Megregor JA, French JI. Chlamydia trachomatis infection during pregnancy. Am J Obstet Gynecol, 1991,164:1782-1789.
曹泽毅.中华妇产科学.第二版,北京:人民卫生出版社,2000:345.
Lan J, Melgers I, CJLM, et al. Prevalence and serovar distribution of asymptomatic cervical Chlamydia trachomatis infections as determined by highly sensitive PCR. J Clin Microbiol 1995,33(12): 3194-3197.
Mardin AD, Su H, Klein MH, et al. Poliovirus hybrids expressing neutralization epitopes from variable domains I and Ⅳ of the major outer membrane protein of Chlamydia trachomatis elicit broadly cross-reactive C. trachomatis-neutralizing antibodies, antibodies, antibodies, Infect Immun 1995; 63(3): 1116-1121.
An Q, Radcliffe G, Vassallo R, et al. Infection With a plasmid-free variant
chlamydia related to Chlamydia trachomatis identified by using multiple assay for nucleic acid detection. J Clin Microbiol 1992,30(11): 2814-2821.
Rodriguez P, Vekris A, Barbeyrac BD, et al. Typing of Chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene. J Clin Microbiol 1991,29(6): 1132-1136.
Frost E, Deslandes S, Bowgaux-Ramoisy D. Chlamydia trachomatis serovars in 435 urogenital specimens typed by restriction endonuclease analysis of amplified DNA, JID 1993,168(8)497-501.
余加林，吴仕孝.连接酶链反应在沙眼衣原体感染诊断中的应用.重庆医科大学学报.1996,21(6): 409-412.
Andrews WW, Goldenberg RL, Mercer B, et al. The Preterm Prediction Study: association of second-trimester genitourinary Chlamydia trachomatis infection with subsequent spontaneous prêt, rm birth. Am J Obstet Gynecol 2000; 183:662-668.
Poole E, Lamont. Chlamydia trachomatis serovar differentiations by direct sequence analysis of the variable segment 4 region of the major outer membrane protein. Infect Immune 1992; 60(3): 1089.
Schachter J, Stamm W, Chernesky MA, et al. Nonculture tests for genital tract Chlamydial infection. Sex Tran Dis 1992; 19:243-244.
Lebar WD. Keeping up with new technology: new approaches to diagnosis of Chlamydial infection. Clin Chem 1996,42(5): 809-812.
Kessler HH, Pierer K, Stuenzner D, et al. Rapid detection of Chlamydial trachomatis in conjunctival, pharyngeal, and urethral specimens with a new polymerase chain reaction assay. Sex Tran Dis 1994,21(4): 191-195.
俞信忠,许平，张国荣.绍兴地区临产前孕妇沙眼衣原体感染的调查.中国优生与遗传杂志,1999,7(1):30.
陈仕红,妊娠期妇女沙眼衣原体感染的检测报告.中国优生与遗传杂志,1999,7(4):28.
苏亦平,花玉波,金红,等.延边地区孕妇沙眼衣原体感染研究.中国实验诊断学,1998,2(3):121.
应长青,王滨有,郑冬梅,等.孕妇沙眼衣原体感染与妊娠结局及新生儿发病的关系.中华妇产科杂志,1999,34(6):384-350.
张健,卢静,孕妇沙眼衣原体感染与新生儿感染关系的研究.济宁医学院学
报,2002,25(1):55-56.
童皎,围产期沙眼衣原体感染研究.新生儿科杂志,2003,18(4):148-150.
黄艳丽,沙眼衣原体感染与妊娠结局的临床观察.中华医院感染学杂志,2001,11(2):111.
李玉梅,鲁继荣,傅文永,等. 沙眼衣原体垂直传播基因水平的快速检测.临床儿科杂志,2000,18(3):133-134.
鲁继荣, 李玉梅, 傅文永,等. 沙眼衣原体母婴垂直传播的研究.中华儿科杂志,1999,37(12):739-741.
张春平,朱道银,郭晓霞.沙眼衣原体子宫内感染途径的研究.中华妇产科杂志,2002,37(3):149-151.
吴仕孝,沈犁,刘官信.母婴垂直传播沙眼衣原体多聚酶链反应及测序研究.中华儿科杂志,1997,35(6):283-285.
Pao CC, Kao SM, Wang HC, et al. Intraumniotic of Chlamydia trachomatis deoxyribonucleic acid sequences by polymerase chain reaction. Am J Obstet Gynecol, 1991, 164: 1295-1299.
丁瑛,顾惠珍,龚增娥,等. 孕妇及新生儿沙眼衣原体的临床观察.中华妇产科杂志,1995,30(2):74-76.
Megregor JA, French JI. Chlamydia trachomatis infection during pregnancy. Am J Obstet Gynecol, 1991,164:1782-1789.
曹泽毅.中华妇产科学.第二版,北京:人民卫生出版社,2000:345.
Lan J, Melgers I, CJLM, et al. Prevalence and serovar distribution of asymptomatic cervical Chlamydia trachomatis infections as determined by highly sensitive PCR. J Clin Microbiol 1995,33(12): 3194-3197.
Mardin AD, Su H, Klein MH, et al. Poliovirus hybrids expressing neutralization epitopes from variable domains I and Ⅳ of the major outer membrane protein of Chlamydia trachomatis elicit broadly cross-reactive C. trachomatis-neutralizing antibodies, antibodies, antibodies, Infect Immun 1995; 63(3): 1116-1121.
An Q, Radcliffe G, Vassallo R, et al. Infection With a plasmid-free variant
chlamydia related to Chlamydia trachomatis identified by using multiple assay for nucleic acid detection. J Clin Microbiol 1992,30(11): 2814-2821.
Rodriguez P, Vekris A, Barbeyrac BD, et al. Typing of Chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene. J Clin Microbiol 1991,29(6): 1132-1136.
Frost E, Deslandes S, Bowgaux-Ramoisy D. Chlamydia trachomatis serovars in 435 urogenital specimens typed by restriction endonuclease analysis of amplified DNA, JID 1993,168(8)497-501.
余加林，吴仕孝.连接酶链反应在沙眼衣原体感染诊断中的应用.重庆医科大学学报.1996,21(6): 409-412.