Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples

Objective: To apply the polymerase chain reaction (PCR) to serum samples for the rapid diagnosis of Legionnaire's disease using the L5SL9 and L5SR93 primers designed to generate a 104-base-pair (bp) fragment from the 5S RNA gene of Legionella spp. The amplified product was detected by electrophoresis and by hybridization with the L5S–1-specific probe.
Methods: Single specimens of serum obtained from 24 patients with confirmed legionellosis, at different stages of their disease, were tested by PCR. Additionally, 10 serum samples from patients with no clinical symptoms of pneumonia and 10 samples from patients suffering from pneumonia caused by Mycoplasma pneumoniae, Coxiella bumetii or Chlamydia psittaci were also tested as controls in order to determine the specificity of the method.
Results: Of the 24 examined serum samples, the amplified products from 12 hybridized with the L5S–1 probe (sensitivity 50%). None of the negative controls was positive after PCR. No correlation was found between the day of illness and the positivity in the test.
Conclusions: The PCR technique could be applied as a diagnostic tool for the rapid diagnosis of legionellosis in serum samples after modification, mainly to improve its sensitivity.     

Using the polymerase chain reaction to maintain DNA probe inventories in clinical and diagnostic laboratories

Clinical and diagnostic DNA laboratories must maintain a large inventory of DNA probes for use in hybridization studies. The preparation of plasmid DNA and isolation of DNA fragments for use as probes in both expensive and time consuming. We present here a rapid and relatively inexpensive method of producing large amounts of DNA fragments from stocks, using the polymerase chain reaction (PCR). Our experience over the past year using this technique has been very positive and we believe many laboratories could benefit by employing such a labor-saving approach to maintaining DNA probes. The technique uses the bacteriophage M13 DNA sequencing primers to amplify cloned inserts contained in commonly used plasmid vectors. As examples, we illustrate the use of DNA produced in this manner as probes for linkage analysis of the fragile X syndrome and for detection of deletions in the Duchenne muscular dystrophy gene. We have also found that at least two probes can be amplified in the same PCR reaction, allowing the detection of two different restriction fragment length polymorphisms (RFLP) simultaneously. It should be possible for laboratories to devise strategies particular to their individual needs using more than one DNA probe produced in the same PCR reaction to detect RFLP's. Such strategies would need only to consider that the predicted alleles of the multiple polymorphisms do not migrate to the same position during electrophoresis. Stocks of single or multiple probes produced by the PCR could then be maintained for more rapid Southern analyses.     

解脲脲原体套式（Nested）PCR检测研究

本文报告解脲脲原体（ＵＵ）的套式（ＮＥＳＴＥＤ）ＰＣＲ检测方法。经方法学考核表明，本法的特异性、灵敏度以及试剂的稳定性和对临检标本的顺应性均较好。９３份各种生殖道炎症患者之宫颈拭子标本套式ＰＣＲ检出２１份阳性（阳性率２２．６％），而市售ＰＣＲ试剂盒仅检出１份阳性（阳性率１．１％）。前者阳性检出率明显高于后者（Ｐ＜０．０１）。     

Evaluation of arbitrarily primed polymerase chain reaction analysis for typing Legionella pneumophila

Objective: To evaluate the performance of arbitrarily primed polymerase chain reaction (AP-PCR) analysis in epidemiologic typing of Legionella pneumophila.
Methods: Sixty-two isolates of L. pneumophila of serogroups 1, 3, 6 and 10, including epidemiologically related and unrelated isolates, were analyzed by AP-PCR using the primer BG2. Twenty-six of the serogroup 1 isolates were typed by pulsed-field gel electrophoresis (PFGE).
Results: AP-PCR analysis showed 98% typeability and complete reproducibility. A majority of unrelated isolates of each serogroup could be distinguished (discrimination index: 92%). Clinical isolates showed AP-PCR patterns indistinguishable from those of the isolates of the related environmental source. PFGE and AP-PCR results were in agreement for 88% of isolates.
Conclusions: Single-primer AP-PCR analysis can be used as a simple and reproducible screening method for typing L. pneumophila strains of different serogroups.     

Direct detection of Mycobacterium tuberculosis in respiratory samples from patients in Scandinavia by polymerase chain reaction

Objective: To investigate the use of DNA amplification by the polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis directly in human respiratory specimens.
Methods: The PCR assay employed was the Amplicor M. tuberculosis Test (Roche Diagnostics, Switzerland), which uses the 16S rDNA as the target template. Nine hundred and sixty samples from 741 patients in two clinical microbiology laboratories in Norway and Sweden were processed by routine culture analysis and PCR.
Results: Of the 56 specimens containing cultivatable M. tuberculosis , 49 (87.5%) were detected by PCR. Among the 904 culture-negative specimens, 897 samples were negative also by PCR and seven (0.8%) were positive by PCR. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of PCR were 91.7%, 99.6%, 94.2% and 99.4% for laboratory 1 and 80.0%, 98.7%, 76.2% and 99.0% for laboratory 2, respectively. For both laboratories combined the values were 87.5%, 99.2%, 87.5% and 99.2%.
Conclusions: These results indicate that multiple (two or three) respiratory samples from each patient should be tested, to allow sufficient accuracy in detecting M. tuberculosis in the specimens. Still, the labor-intensive format of this test necessitates strong clinical indications and patient prioritization to provide a service feasible within the current limits of routine laboratories.     

Standardization of fungal polymerase chain reaction for the early diagnosis of invasive fungal infection

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10⁶–10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.     

肺炎衣原体的套式（Nested）PCR检测及其临床意义

肺炎衣原体（C_(pn)）可致上、下呼吸道的炎症，并且是非典型性肺炎综合征（APS）的主要病原体之一。C_(pn)已占肺炎病原体的第三或第四位。近年的深入研究表明，C_(pn)还能引起呼吸道之外的脏器炎症。由于C_(pn)为专性细胞内寄生菌，因此培养困难。目前国内尚缺乏C_(pn)感染诊断方法。本文介绍一种灵敏、特异、简便的C_(pn)套式PCR检测方法。应用本法检测55例呼吸道感染者之咽拭子，共检出9例阳性（阳性率16．2％）。表明C_(pn)的呼吸道感染在我国并不少见。C_(pn)套式PCR方法的建立，对于我国的C_(pn)人群感染率和C_(pn)感染的分子流行学研究有着极其重要的意义。     

Identification and typing of molluscum contagiosum virus in clinical specimens by polymerase chain reaction

A polymerase chain reaction (PCR) which enables the detection of molluscum contagiosum virus (MCV) genomes in either fresh or formalin-fixed clinical specimens is described. The primers used were designed to amplify a 167 bp region of the 3.8kbp HindIII fragment K of the MCV 1 genome. The ability of this PCR to detect three common MCV types (1, 1v and 2) in clini-cal specimens was confirmed using frozen extracts from 75 molluscum lesions, and digests of single sections of 11 formalin-fixed, paraf-fin-embedded lesions; all of which had been previously typed by Southern hybridisation. In addition, 2 specimens previously negative by hybridisation were shown to be positive for MCV DNA by PCR. Confirmation of the identity of the PCR products and distinction between the two major MCV types (MCV 1/1v versus MCV 2) was achieved by comparison of the results of cleavage with the restriction endonucleases HhaI and SacI. Sequencing of the PCR products revealed complete homology between MCV 1 and 1v, but minor nucleotide variations between MCV 1/1v and MCV 2 were identified. As well as providing a highly sensitive means of diagnosis, the technique may also prove useful for investigations into the pathogenesis, epidemiology and natural history of molluscum contagiosum infection. J. Med. Virol. 53:205–211, 1997. © 1997 Wiley-Liss, Inc.     

Rapid,sensitive, specific,and quantitative detection of human T-cell leukemia virus type 1 sequence in peripheral blood mononuclear cells by an improved polymerase chain reaction method with nested primers

Improving on the nested double polymerase chain reaction (PCR) described previously, we have developed a new two-step PCR (TS-PCR) method for detecting more specifically the human T-cell leukemia virus type 1 (HTLV-1) proviral sequences in peripheral blood mononuclear cells (PBMC). In our TS-PCR method, the point of modification is to use optimal concentrations of primers in the first amplification step in the range of 0.01–0.025 µM. This increases sensitivity and specificity enough to detect from 1 to 10⁵ copies of template DNA without radioisotopes. This method is rapid because of completion in 1 day and is also applicable for quantitative detection of clinical specimens. The data show that the quantitative detection of HTLV-1 proviral sequences by this method correlates with the anti-HTLV-1 antibody titers from serologic analysis of seropositive healthy carriers. Moreover, the TS-PCR method using each specific primer was also attempted for successful detection of other viral genomes; therefore, the principle of this method is widely suitable for routine detection of genomes in the basic and clinical microbiological fields.     

Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia

Objective   To study the clinical significance of a nested polymerase chain reaction (PCR) method compared to immunofluorescence (IF) for detection of Pneumocystis carinii .
Methods   The medical records of 89 patients with 91 episodes of pneumonia were scrutinised retrospectively. The pneumonia episodes were divided into categories according to the likelihood that the patient had had clinical Pneumocystis carinii pneumonia (PCP). All respiratory tract samples from the 89 patients (34 broncho-alveolar lavage (BAL) and 57 sputa) were tested for Pneumocystis carinii by IF and nested PCR.
Results   Fifteen episodes, as diagnosed by IF, were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 60%, specificity 97%). Among the P. carinii DNA-positive episodes, detected with nested PCR, 24 were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 96%, specificity 59%), since all IF-positive samples were nested PCR positive. Only one pneumonia episode classified as a probable PCP, was negative with both methods, as applied to a BAL sample.
Conclusions   IF applied to BAL or sputum seems to be the most specific method for diagnosis of clinical PCP. Additional clinical cases can be found by nested PCR, although this then gives a high risk of detecting subclinical colonisation of P. carinii .     

聚合酶链反应检测淋菌的结果分析

本文应用聚合酶链反应(PCR)技术对3853例疑为淋菌感染患者进行了检测。检出阳性者961例，阳性率24．9％，对照培养法576例．检出阳性者38例，阳性率6．6％。结果表明PCR法具有高度特异性、敏感性和选择性．且快速、简便．是临床处理大批标本的一种最理想的方法．适于临床推广。     

应用巢式PCR对单细胞进行性别诊断的初步研究

目的：应用巢式PCR技术对人类植入前胚胎进行性别诊断。方法：收集单个或两个淋巴细胞和卵裂球（50个／组），按不同的方法处理单细胞（纯水法、冻融法、碱法），而后行巢式PCR扩增牙釉质基因。结果：纯水法、冻融法、碱法处理后，扩增率分别为83％、94％、95％。后两种处理方法的扩增效率明显高于纯水法（P＜0．01），通过检测正常男性单淋巴细胞基因型发现3种方法等位基因脱失率分别为24％、12％、4％，差异有显著性（P＜0．05）。两个淋巴细胞或卵裂球的扩增率及等位基因脱失率与单细胞相比差异无显著性。结论：提高植入前遗传学诊断的准确性主要取决于如何克服单细胞PCR的缺点，采用碱法裂解单细胞及取两个卵裂球进行检测可提高用于性别诊断的单细胞PCR技术准确性和敏感性。     

Direct detection of verotoxin genes in stool samples by polymerase chain reaction in hemolytic uremic syndrome patients in France

Objective: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients.
Method: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS.
Results: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene. VTEC was isolated from only 32 of the 51 PCR-positive samples. E. coli O157:H7 was isolated from five patients. E. coli O111 was isolated from seven patients during an outbreak of HUS. Among the other VT2 E. coli strains, only four were serotypable. Of the 51 PCR-positive stools, 19 were culture negative for VTEC.
Conclusions: This study provides evidence that in France E. coli O157 and other VTEC serotypes are involved in HUS.     

The polymerase chain reaction in histopathology

Thanks to the advent of the polymerase chain reaction (PCR) molecular genetic study of histological samples is now a relatively straightforward task and the vast histopathology archives are now open to molecular analysis. In this review we outline technical aspects of PCR analysis of histological material and evaluate its application to the diagnosis and study of genetic, infectious and neoplastic disease. In addition, we describe a number of newly developed methods for the correlation of PCR analysis with histology, which will aid the understanding of the molecular basis of pathological processes.     

Comparison of in-house polymerase chain reaction method with the Roche Amplicor technique for detection of Mycobacterium tuberculosis in cytological specimens

Two techniques have been approved by the United States FDA for diagnosis of tuberculosis in smear positive sputa: LCX M. tuberculosis, a ligase chain reaction procedure manufactured by Abbott Laboratories, and Amplicor, a polymerase chain reaction (PCR) procedure manufactured by Roche. However, these commercial methods are expensive and beyond the reach of laboratories in most developing countries. We compared the Roche Amplicor kit with an in-house PCR using a primer set for Mycobacterium tuberculosis/bovis directed at MPB 64 protein gene. It was able to distinguish between M. tuberculosis, M. avium, M. gordonii, M. intracellularae, and M. kansasii. Fifty-seven cytological samples were submitted to the laboratory for molecular diagnosis of M. tuberculosis. Both procedures were run on every sample submitted and the two methods agreed completely. The custom-made method is less expensive than the commercial technique.     

Evaluation of the polymerase chain reaction for the detection of human papillomavirus from urine

The ability to detect the presence of human pap-illomavirus (HPV)-DNA sequences in urine was evaluated using polymerase chain reaction (PCR). DMA was purified and extracted from urine samples, and subjected to 40 cycles of amplification using the consensus primer pair MY11 and MY09. Coamplification using the β-globin primers, GH20 and PC04, was performed as an internal reaction control. Following assay optimization, urine samples from 22 women undergoing examination for cervical dysplasia were tested for the presence of HPV-DNA. PCR assay results were correlated with cytologic and histo-logic findings as well as Vira Type(tm) assay results. Overall, HPV was detected by PCR in 16 (76%) of the interpretable samples. HPV sequences were detected in 13 (87%) of the 15 specimens from women showing evidence of condylomata, dysplasia, or invasive carcinoma. HPV was detected in 3 (50%) of the women whose cytologic or his-tologic results were either negative or showed benign atypia. Although the sample size in this study is small, our results show that HPV can be detected by PCR in a majority of individuals showing evidence of HPV infection. The method described provides a means for the clinical laboratory to detect a broad range of HPV types from using a sample obtained by noninvasive techniques. The ability to easily obtain urine would allow for increased numbers of individuals to be tested, and thus, aid in our understanding of HPV. © 1995 Wiley-Liss, Inc.