新生儿肺炎患儿人型支原体套式PCR检测研究

本文应用套式ＰＣＲ检测了１５６例新生儿肺炎患儿鼻咽部分泌物中的人型支原体特异性ＤＮＡ，其结果：患儿阳性数１３０例，阳性率８３．３％，而应用人型支原体培养法及单式ＰＣＲ阳性率均比应用套式ＰＣＲ为低，提示了临床必须对人型支原体的感染率及致病性引起足够的重视。同时，通过本文研究也提示了临床对于不同的支原体感染可采用不同的抗生素治疗。目前国内对于人型支原体与新生儿肺炎的相关性报道尚未检索到。     

聚合酶链反应在小儿支原体肺炎诊断中的应用价值

为探讨ＰＣＲ在小儿支原体肺炎诊断中的实际应用价值，对４７例临床疑诊支原体肺炎的病儿做了咽拭子ＰＣＲ－ＭＰ测定，同时做了血冷凝集试验（ＣＡＴ）。结果：ＰＣＲ－ＭＰ阳性２１例，真阳性１５例，假阴性１例，敏感度为９３．８％。假阳性６例，占检测标本的１２．８％，占检测报告阳性总数的２８．６％，ＣＡＴ阳性１２例。确诊支原体肺炎１６例，其中１５例经ＰＣＲ早期确诊。结论：ＰＣＲ－ＭＰ测定有利于支原体肺炎的早期诊     

急性白血病患儿脑脊液PCR扩增HCMV－DNA的研究

应用多聚酶链式反应（ＰＣＲ）扩增和洋地黄探针杂交的方法，对５３例急性白血病（ＡＬ）患儿脑脊液（ＣＳＦ）样本进行了检测。经过ＰＣＲ扩增，可见明显的人巨细胞病毒ＤＮＡ（ＨＣＭＶ－ＤＮＡ）４００ｂｐ扩增带；经洋地黄探针特异性杂交鉴定，ＡＬ患儿ＣＳＦ标本ＰＣＲ扩增产物的ＨＣＭＶ－ＤＮＡ阳性检出率为８３．０２％。本实验为ＡＬ病人ＣＳＦ的ＨＣＭＶ检测提供了一个简便方法。     

急性白血病患儿脑脊液PCR扩增HCMV—DNA的研究

应用多聚酶链式反应（ＰＣＲ）扩增和洋地黄深针杂交的方法，对５３例急性白血病（ＡＬ）患儿脑脊液（ＣＳＦ）样本进行了检测，经过ＰＣＲ扩增，可见明显的人巨细胞病毒ＤＮＡ（ＨＣＭＶ－ＤＮＡ）４００ｂｐ扩增带，经洋地黄探针特异性杂交鉴定，ＡＬ患儿ＣＳＦ标本ＰＣＲ扩增产生的ＨＣＭＶ－ＤＮＡ阳性检出率为８３．０２％。本实验为ＡＬ病人ＣＳＦ的ＨＣＭＶ检测提供了一个简便方法。     

用PCR技术检测喘息性疾病患儿肺炎支原体的研究

本文应用聚合酶链反应（ＰＣＲ）技术检测了１５６例喘息性疾病患儿咽拭子标本的肺炎支原体（ＭＰ），阳性率为１２．２％，对照组为同期３８例肺炎患儿的咽拭子标本，ＭＰ阳性率为１７．３％，二组ＭＰ感染率无显著性差异（ｘ２＝１．７７，Ｐ＞０．０５）。提示：ＭＰ不仅是肺炎也是喘息性疾病患儿常见的致病微生物。因此，对喘息性疾病患儿应常规用ＰＣＲ技术检测ＭＰ，以提高诊断和治疗水平     

新生儿包涵体结膜炎CT-nPCR检测的分析

本文对８５例新生儿结膜炎患儿进行沙眼衣原体套式ＰＣＲ（ＣＴ－ｎｅｓｔｅｄＰＣＲ）进行检测。２１例ＣＴ检测阳性（２４．９％）， 时对其母亲生殖道分泌物也做了检测，结果均为ＣＴ阳性。其中２０例为阴道分娩，１例剖宫产。说明新生儿包涵体结膜炎为母婴传播性疾病。孕期筛查ＣＴ，并给以有效治疗，减少新生儿患病率，达到优生优育的目的。     

多聚酶链反应技术在NHL基因诊断中的应用研究

收集非何杰金淋巴瘤（ＮＨＬ）标本５９例。用全Ｔ（ＵＣＨＬ一１）与全Ｂ（Ｌ２６）ＭｃＡｂ作免疫组化分型。然后应用ｌｇＨ单轮和半重叠基因引物，Ｔ细胞受体β（ＴＣＲ＿β）基因引物进行多聚酶链反应（ＰＣＲ）扩增检测克隆性基因重排。检测阳性结果：新鲜组织ｌｇＨ７１．４％（１０．／１４），ＴＣＲ＿β８３．３％（１０／１２）。石蜡包埋组织ＩｇＨ（半重叠扩增）８０％（１２／１５），ＴＣＲ＿β７３．３％（１１／１５）。无假阳性。其中有６例有争议的疑难病例明确了诊断。结果表明ＰＣＲ技术是当前最特异、敏感而快速的ＮＨＬ克隆性基因重排检测方法。     

套式PCR鉴定精液解脲脲原体培养结果报告

应用套式（Ｎｅｓｔｅｄ）ＰＣＲ鉴定７０例精液解脲脲原体培养结果。显示两种方法的阳性符合率为７８．０４％，其中包括：１．培养结果２４－４８小时阳性，而后转阴性１３例，经套式ＰＣＲ检测６例阳性，阳性率为４６．１％，占阳性经率的１６．２％（６／３７）。２．培养７２小时后才显示阳性结果６例，经套式ＰＣＲ检测均为阳性，占阳性比率的１６．２％（６／３７），说明培养法只能用于初筛。     

鼻咽癌组织中人疱疹病毒6型感染及其临床意义

目的　探讨人疱疹病毒６ 型（ＨＨＶ６） 与鼻咽癌（ＮＰＣ） 发病的关系。方法　采用聚合酶链反应（ＰＣＲ） 和原位杂交同时检测正常鼻咽组织和ＮＰＣ 癌前鼻咽组织以及ＮＰＣ 组织中ＨＨＶ６ 和ＥＢＶＤＮＡ 的存在情况。采用免疫组化检测３６ 例ＮＰＣ 组织的ＥＢＶＬＭＰ１ 蛋白表达。结果　在４２ 例ＮＰＣ组织、２１ 例鼻咽癌前病变组织中,经ＰＣＲ检出ＨＨＶ６ ＤＮＡ,阳性率分别为３０－９％ （１３ 例） 和４－７ ％（１ 例） 。ＥＢＶＤＮＡ为９５－２％ （４０ 例） 和６６－６ ％ （１４ 例）。２７ 例正常鼻咽组织未检出ＨＨＶ６ ＤＮＡ,ＥＢＶＤＮＡ为２５－９％ （７ 例）。经ＩＳＨ,仅在１３ 例ＰＣＲＨＨＶ６ ＤＮＡ阳性的ＮＰＣ组织中检出１１ 例阳性,３６ 例ＮＰＣ组织、１０ 例鼻咽癌前病变组织ＩＳＨＥＢＶＤＮＡ 阳性。ＮＰＣ组织的ＥＢＶＬＭＰ１ 蛋白阳性率为４７－２ ％（１７３６）。结论　鼻咽癌组织中存在ＨＨＶ６ 感染,提示ＨＨＶ６ 感染与ＮＰＣ有关。ＨＨＶ６ 感染与ＥＢ病毒ＬＭＰ１ 蛋白表达上调呈正相关,提示在ＮＰＣ 的发生中ＨＨＶ６ 可能直接参与和（ 或） 通过上调ＥＢＶＬＭＰ１ 的表达而起一定作用     

宫颈感染解脲支原体与不良妊娠结局的关系

目的：探讨宫颈解脲支原体（ＵＵ）在不同状态下与不良妊娠结局的关系。方法：通过聚合酶链反应（ＰＣＲ）对２１０例孕发的宫颈分泌物进行ｕｕＤＮＡ检测，用酶联免疫吸附试验（ＥＬＩＳＡ）检测孕妇血清抗ｕｕＩｇＭ以两项检测结果同时阳性做为宫颈ｕｕ感染的标准，以ｕｕＤＮＡ阳性而血清抗ｕｕＩｇ阴性作为宫颈ｕｕ的携带状态的标准。结果：２１０例孕妇中宫发泌物ｕｕＤＮＡ阳性１００例，占４７．６％，宫颈分泌物ｕｕＤＮＡ及     

Detection of enterotoxigenic Bacteroides fragilis by PCR.

Strains of enterotoxigenic Bacteroides fragilis (ETBF) are associated with diarrhea in young farm animals and, at least in particular settings, in children. Enterotoxin production by ETBF is currently detected by a tissue culture assay with HT-29 cells. We have developed a PCR assay based on the detection of the enterotoxin gene to identify ETBF in culture and in stool samples. Overall, 113 bacterial strains were examined, including 3 B. fragilis reference strains, 75 B. fragilis isolates (comprising 40 ETBF isolates), 20 Bacteroides spp. other than B. fragilis, and 15 strains belonging to other genera. Complete agreement was found between the results of the tissue culture assay and those of the PCR for our strains. PCR was also used to detect ETBF directly in fecal samples. Stools from two healthy volunteers were spiked with known numbers of ETBF and were processed by three different methods. A culture method, which required inoculation of the stools on selective plates and the collection of the whole bacterial growth ("sweeps"), was found to be the most sensitive. PCR performed with the plate sweeps yielded amplification products with a detection limit of 10(5) to 10(4) CFU/g of feces. By this method 18 samples of diarrheic stools (10 positive and 8 negative for ETBF) were examined. The results of the PCR were in accordance with the culture results in all cases. The proposed PCR assay represents a diagnostic tool for the rapid identification of ETBF in culture as well as in fecal samples.     

Detection of Bacteroides fragilis in clinical specimens by PCR.

The direct detection of Bacteroides fragilis from clinical specimens was examined by using the PCR method for amplifying a specific fragment of the glutamine synthetase gene from B. fragilis. By this method, all five B. fragilis strains tested were detected, but DNAs from anaerobic bacteria of 24 other species tested, from aerobic bacteria of 12 species tested, and from human leukocytes were not amplified. Using the nested PCR method, we were able to detect as little as one bacterial cell or 100 fg of chromosomal DNA of B. fragilis. A total of 39 clinical specimens, which consisted of 19 bronchial aspirates, 10 percutaneous lung aspirates, 2 transtracheal aspirates, 6 pleural fluid specimens, and 2 pus specimens, were tested. All four culture-positive samples, of which two were bronchial aspirates, one was pleural fluid, and one was pus, were positive by PCR. Among 35 culture-negative samples, 2 bronchial aspirates were positive by PCR. One was from a patient whose two previous samples were positive by both culture and PCR. It had been submitted for culture several hours after collection, and clindamycin had been administered to the patient before collection of the specimen. The other bronchial aspirate positive by PCR was from a pneumonia patient who had also been administered clindamycin. We believe that B. fragilis was present in these two specimens but that either it was dead, it was below the level detectable by culture, or the process of anaerobic culture was unsuccessful. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobes in clinical specimens.     

Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.     

High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR.

PCR was used to detect Haemophilus influenzae in samples of nasopharyngeal secretion and middle ear effusion (MEE). Nasopharyngeal secretions were collected from 102 patients with otitis media with effusion and from 111 healthy subjects. Eighty samples of MEE were collected from patients with otitis media with effusion. A pair of primers was designed to amplify a DNA segment of the gene encoding P6 outer membrane protein of H. influenzae. The amplified PCR product was detected with an internal probe that hybridized specifically to the P6 DNA of H. influenzae. Samples of MEE and nasopharyngeal secretion were also examined by a conventional culture method. The incidence of P6 gene DNA in nasopharyngeal secretions detected by PCR was about two times higher than that of H. influenzae detected by the conventional culture. Culture-positive samples were all positive in the PCR test. In MEEs, the rate of detection of the P6 gene DNA target was about five times higher than that of H. influenzae detected by the culture method. All patients who had P6 gene DNA in MEEs were found to have the DNA in nasopharyngeal secretions. These findings suggest that the presence of H. influenzae in MEEs and in nasopharyngeal secretions is more common than previously reported.     

Detection of Mycoplasma pneumoniae in clinical samples from pediatric patients by polymerase chain reaction.

The polymerase chain reaction (PCR) technique was used to detect Mycoplasma pneumoniae DNA in clinical samples (nasopharyngeal aspirations or bronchoalveolar lavages) obtained from 100 children, 1 month to 16 years old. PCR allowed the detection of M. pneumoniae DNA from 20 out of the 100 patients studied. In 16 cases, PCR positivity was associated with acute respiratory symptomatology. For five PCR-positive patients, a positive culture or a serological response evidenced acute M. pneumoniae infections. A lack of antibody response was observed particularly with immunocompromised children and infants less than 12 months old. The amount of M. pneumoniae DNA in the PCR was estimated in a semiquantitative way by comparison of its hybridization signal with those obtained for 100, 10, and 1 color-changing unit (CCU) of the M. pneumoniae FH strain. Small amounts (less than or equal to 10(2) CCU/ml) of M. pneumoniae were found in samples from asymptomatic patients, while larger amounts (greater than or equal to 10(2) to greater than or equal to 10(4) CCU/ml) were found for 8 out of 10 patients with acute pneumonia.     

Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation

Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive for Legionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionella species by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.     

Use of PCR to identify enteroaggregative Escherichia coli as an important cause of acute diarrhoea among children living in Calcutta, India.

The importance of enteroaggregative Escherichia coli (EAggEC) as a possible aetiological agent of acute diarrhoea among children in Calcutta, India, was investigated. Simultaneously the use of a previously described PCR diagnostic system was assessed for its ability to identify EAggEC infection. E. coli strains isolated during a 1-year case-control study from faecal samples of 388 children aged <5 years, with or without diarrhoea, were examined for EAggEC by HeLa cell adherence assay in parallel with a PCR assay with primers generated from an EAggEC DNA probe. A blind comparison was made between the two methods to determine their diagnostic potential. E. coli isolates that adhered to HeLa cells in an aggregative pattern were the sole isolates significantly more often in 254 cases (9%) than in 134 control (2%) children. Age stratification showed that EAggEC were isolated more frequently from children aged <36 months. The EAggEC isolates belonged to several O serogroups and showed multiple drug resistance. Both methods were positive for 26 samples, nine samples were positive by PCR alone and seven samples were positive by culture alone, thus indicating a 78% sensitivity and 97% specificity for the PCR assay. EAggEC is an important aetiological agent of acute diarrhoea among infants in and around Calcutta, and the PCR diagnostic system may be useful to identify such infection in epidemiological studies.     

Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification

Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.