The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae

A DNA probe encoding approximately 80% of the 18-kDa protein gene of Mycobacterium leprae was isolated and tested for specificity by assessing hybridization of the probe to genomic DNA from taxonomically related and unrelated DNA samples. The 360-base-pair (bp) probe was specific for M. leprae DNA and did not hybridize with genomic DNA from 18 species of bacteria nor with DNA from human, murine, and armadillo sources. Oligonucleotide primers were synthesized corresponding to the 5' and 3' ends of the 360-bp fragment to yield a fragment of similar size on amplification of M. leprae DNA by the polymerase chain reaction (PCR). A simple procedure for DNA extraction from M. leprae-infected tissues was developed that provided suitable template DNA for amplification. The PCR test was specific for M. leprae DNA from human and murine sources and detected M. leprae DNA in biopsies from leprosy patients and from control and uninfected human skin biopsy preparations seeded with as few as 100 M. leprae.     

Sensitive detection of Pneumocystis jirovecii DNA in gastric wash using nested polymerase chain reaction

One hundred and five samples of gastric washes were obtained from 52 pediatric patients. Eleven of the 105 samples (10%) gave positive results using immunofluorescence antibody test (IFA) for Pneumocystis jirovecii. Single-step polymerase chain reaction (PCR) produced 13% (14 samples), whereas detection by nested PCR was increased to 65 samples (62%). Moderate agreement (kappa = 0.5) was found between test results of IFA and single-step PCR, but no agreement was found between the results of IFA and nested PCR (kappa = 0.1).     

DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction.

The objective of this project was to construct specific and sensitive molecular probes and amplification primers for Cryptosporidium parvum that could be used in diagnosis, retrospective tissue studies, and in epidemiologic surveys. Whole genomic DNA was extracted from oocysts of C. parvum purified from human and bovine feces. A genomic library was constructed in plasmid pUC18 and propagated in Escherichia coli DH5 alpha. Transformants were screened by colony hybridization and autoradiography. The 2.3-kilobase segment in plasmid pHC1, a clone specific for C. parvum, was sequenced by the Sanger method. Computer analysis gave a G+C content of 35%. A 400-base region (bases 470-870) was selected as an amplification target because it contained a unique restriction endonuclease site that could serve as a useful marker. Primers of 26 nucleotides each were synthesized. Sensitive and specific amplification of the target sequence was demonstrated both by ethidium bromide staining of agarose and acrylamide gels, and by hybridization with chemiluminescence-labeled synthetic oligonucleotide probes.     

Sensitivity and specificity of polymerase chain reaction for the detection of Toxoplasma gondii DNA added to laboratory samples

We studied the sensitivity and specificity of PCR to detect T. gondii DNA by aliquoting various concentration of tachyzoites into laboratory specimens from 60 positive and 10 negative buffy-coat samples. We were able to detect the specific gene from purified DNA samples containing as few as 0.25 parasites per 100,000 human leukocytes. These results had an impressive initial 100% specificity but later it decreased because of false-negative data.     

Colorimetric detection of specific DNA segments amplified by polymerase chain reactions.

The polymerase chain reaction (PCR) procedure has many potential applications in mass screening. We describe here a general assay for colorimetric detection of amplified DNA. The target DNA is first amplified by PCR, and then a second set of oligonucleotides, nested between the first two, is incorporated by three or more PCR cycles. These oligonucleotides bear ligands: for example, one can be biotinylated and the other can contain a site for a double-stranded DNA-binding protein. After linkage to an immobilized affinity reagent (such as a cloned DNA-binding protein, which we describe here) and labeling with a second affinity reagent (for example, avidin) linked to horseradish peroxidase, reaction with a chromogenic substrate allows detection of the amplified DNA. This amplified DNA assay (ADA) is rapid, is readily applicable to mass screening, and uses routine equipment. We show here that it can be used to detect human immunodeficiency virus sequences specifically against a background of human DNA.     

DNA probe and polymerase chain reaction for detection and identification of mycobacteria]

The DNA probe and polymerase chain reaction (PCR) technique for detection and identification of mycobacteria were compared with the conventional smear and culture method. The results of identification by DNA probe agreed well with those of the biochemical method. Moreover, six percent of Mycobacterium avium complex (MAC) were revealed to be mycobacteria other than MAC by DNA probe. The nested PCR for detection of gene coding protein antigen b of Mycobacterium tuberculosis complex showed excellent specificity and sensitivity. Then we applied this technique to rapid detection of M. tuberculosis in 222 clinical samples. The agreement between nested PCR and the biochemical method was excellent, and 17 cases were diagnosed by only nested PCR in spite of negative results by smear and culture. These cases were unlikely to have yielded false positive results since their clinical features were compatible to tuberculosis. From these data, it was considered that the DNA probe and PCR technique were extremely useful strategies and would contribute to rapid diagnosis of mycobacterial infectious diseases.     

Evaluation of the detection ofBorrelia burgdorferi DNA in urine samples by polymerase chain reaction

Summary It is difficult in some cases to identify an infection caused byBorrelia burgdorferi and to monitor the effect of therapy. Seropositivity will persist even after successful treatment and therefore may suggest ongoing infection. For direct detection ofB. burgdorferi DNA in human urine samples, the polymerase chain reaction (PCR) was evaluated. A published primer system was selected, which amplifies a 259 bp fragment from the gene encoding the 23S rRNA. The lower detection limit of the primer system was 10 fg of extractedB. burgdorferi DNA. Several methods for the pretreatment of urine sample were tested. Of these, the Geneclean® kit (Bio 101, USA) showed the best results. A total of 114 urine samples from 74 patients belonging to three clinical groups was investigated: (i) 51 samples from 26 patients with active Lyme disease, (ii) 36 samples from 27 patients with previous infection but no symptoms at the time the urine was collected, and (iii) 27 samples from 21 seronegative control patients without Lyme disease.B. burgdorferi DNA was detected in 25 urine samples of 17 patients with active disease, whereas 26 samples from this group of patients were negative. Only one asymptomatic case with previous infection showed a positive result, and the urine samples of the patients without Lyme disease were uniformly negative. Two of four patients from whom samples before and directly after onset of therapy were available converted from negative to positive PCR results after initiation of therapy, accompanied by the symptoms of a Jarisch-Herxheimer reaction. It can be concluded from these results that a positive PCR from urine is with high probability an indicator of active Lyme disease. On the other hand, as only 17 of the 26 patients with active infection were positive, a negative PCR result does not exclude active infection.

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Evaluation des Nachweises vonBorrelia burgdorferi-DNA in Urinproben mittels Polymerase-Kettenreaktion

Zusammenfassung In manchen Fällen ist es schwierig, Infektionen mitBorrelia burgdorferi zu diagnostizieren und den Effekt einer Therapie abzuschätzen. Seropositivität bleibt selbst nach erfolgreicher Therapie bestehen und kann deshalb ein weiterbestehendes Infektionsgeschehen vortäuschen. Zum Direktnachweis vonB. burgdorferi-DNA in menschlichen Urinproben wurde das Verfahren der Polymerase-Kettenreaktion (PCR) evaluiert. Zur Anwendung kam ein Primersystem, dessen Zielsequenz auf dem Gen für die 23S rRNA liegt (Schwartz et al., J. Clin. Microbiol. 30: 3082–3088; 1992). Die Nachweisgrenze des Primersystems betrug 10 fg an extrahierter DNA vonB. burgdorferi. Zur Probenvorbereitung von Urinproben wurden mehrere Methoden getestet. Davon erwies sich das Geneclean® Kit (Fa. Bio 101) als am besten geeignet. Damit wurden insgesamt 114 Urinproben von Patienten aus drei klinischen Gruppen untersucht: (i) 51 Proben von 26 Patienten mit aktiver Lyme-Borreliose, (ii) 36 Proben von 27 symptomlosen Patienten mit vorangegangener Infektion, und (iii) 27 Proben von 21 seronegativen Kontrollpatienten ohne Lyme-Borreliose. Mit Hilfe der PCR bei Probenaufbereitung mit Geneclean® gelang ein Nachweis von Borrelien-DNA in 25 Proben von 17 Patienten mit aktiver Erkrankung, während 26 Proben aus dieser Gruppe negativ waren. Ein asymptomatischer Fall zeigte ein positives Resultat, und die Proben der Patienten ohne Lyme-Borreliose waren einheitlich negativ. Zwei von vier Patienten, von denen Urinproben vor und direkt nach Therapie verfügbar waren, zeigten eine Konversion von negativen zu positiven PCR-Resultaten nach Therapiebeginn, was auf einen Erregerzerfall und vermehrte Ausscheidung von Borrelien-DNA schließen läßt. Die Positivität unter Therapie war korreliert mit dem klinischen Auftreten einer Jarisch-Herxheimer-Reaktion. Die vorliegenden Ergebnisse lassen schließen, daß zwar ein negatives PCR-Ergebnis nicht gegen Krankheitsaktivität spricht, da nur 17 der 26 Patienten mit aktiver Erkrankung positive Resultate zeigten, jedoch daß ein positives PCR-Resultat als Hinweis auf eine aktive Infektion gewertet werden kann.

     

聚合酶链反应检测TT病毒核酸的优化条件

目的探讨聚合酶链反应(PCR)扩增TTV核酸的优化条件,初步测定慢性丙型肝炎及慢性非甲-戊,非庚肝炎患者中TT病毒的检出率.方法将标本分别予以4℃保存1wk、室温保存1wk及1wk内将标本于-35℃与室温间反复冻融6次3种储存方法.分别以SDS-蛋白酶K方法(方法1),NP-40法(方法2)及聚乙二醇法提取TTV DNA,分别以套式PCR与单管套式PCR检测TTV.以PCR直接序列分析方法测定扩增产物以验证扩增产物的特异性.结果在10份已知病毒标志均为阴性的血清中,方法1与方法2提取核酸后可在4份血清中检出TTV,方法3提取核酸后的检出率为0.1次PCR、单管PCR与单管套式PCR对10份已知病毒标志均为阴性血清的TTV检出率分别为10%,40%和40%.将2份血清按原血清、10-1～10-10系列稀释后检测,发现,1次PCR、套式PCR和单管套式PCR的检出水平分别为原倍、10-5与10-4及阴性、10-4与10-4.4℃保存1wk、室温保存1wk及反复冻融标本的最低检测稀释度为10-5,10-4,10-3与10-4,10-4,10-3.对一份PCR产物进行序列分析证实为TTV特异性基因.检测31份慢性丙型肝炎及32份慢性非甲-戊,非庚肝炎标本,阳性率分别为29.03%和34.38%.结论建立了一种敏感、快速、特异的TT病毒检测方法.在1wk内,不同的标本储存方法对检测结果的影响不大,TT病毒可能是慢性非甲-戊,非庚肝炎及输血后肝炎的重要致病因子.     

The use of the polymerase chain reaction in haematology

The polymerase chain reaction (PCR) has rapidly become an invaluable technique for the detection, molecular characterisation and clinical management of a wide variety of haematological disorders. PCR provides a rapid method for the generation of large quantities of relatively pure DNA sequences of interest. This has facilitated nucleotide sequence analysis in both normal and pathological haemopoietic populations and has consequently aided the characterisation of normal molecular organisation and of inherited and acquired genetic defects. PCR amplification has enabled the rapid detection of mutant or polymorphic alleles using allele-specific oligonucleotide probes, aiding both antenatal diagnosis and large scale population screening. The extreme sensitivity of detection of rare genetic events has greatly improved the ability to detect minimal residual malignancy and low levels of viral infection. This article describes the theory and practical aspects of PCR gene amplification and reviews its scientific and clinical applications in haematology.     

鼠尿液中弓形虫B1基因实时荧光定量PCR检测方法的建立

根据GenBank中弓形虫B1基因序列,设计1对引物和1条TaqMan探针。从感染弓形虫小鼠尿液提取弓形虫总DNA,先用普通PCR扩增获得目的基因产物,将纯化的回收产物与pMD18-T载体连接,测序证实为目的片段后,制备系列浓度参照品。再优化实时荧光定量PCR反应体系,并对体系的灵敏度、重复性、线性、特异性和参照品稳定性进行评价。其灵敏度为104拷贝/ml;批内变异系数为2.42%,批间变异系数为4.18%,线性为103～107拷贝/ml,特异性为100%,参照品稳定。该法检测鼠尿样弓形虫DNA具有方便、灵敏、特异和重复性好等优点。     

Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction

OBJECTIVE: Molecular biology techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) are routinely used in research for detection of C trachomatis DNA in synovial samples, and these methods are now in use in some clinical laboratories. This study aimed at determining the method best suited to molecular diagnosis of C trachomatis by examining four standard DNA preparation methods using chlamydia spiked synovial tissue and chlamydia infected monocytes. METHODS: Synovial tissue from a chlamydia negative patient with rheumatoid arthritis was spiked with defined numbers of C trachomatis elementary bodies (EB). Purified human peripheral monocytes from normal donors were infected with the organism at a multiplicity of infection 1:1 in vitro and harvested after four days. DNA was prepared from all samples by four methods: (1) QIAmp tissue kit; (2) homogenisation in 65 degrees C phenol; (3) incubation at 97 degrees C; (4) proteinase K digestion at 97 degrees C. DNA from methods 1 and 2 was subjected to PCR using two different primer sets, each targeting the C trachomatis omp1 gene. LCR was done on DNA prepared by each method. RESULTS: In synovial tissue samples spiked with EB, and in monocytes persistently infected with the organism, preparation of template using the QIAmp tissue kit (method 1) and the hot phenol extraction technique (method 2) allowed sensitive detection of C trachomatis DNA. These methods also produced template from both sample types for LCR. DNA prepared by heat denaturation (method 3) allowed only low sensitivity chlamydia detection in LCR and did not work at all for PCR. Proteinase K digestion plus heat denaturation (method 4) gave template that did not allow amplification in either PCR or LCR assays. CONCLUSIONS: The sensitivity of detection for C trachomatis DNA in synovial tissue by PCR and LCR depends strongly on the method used for preparation of the amplification template. LCR targeting the multicopy chlamydial plasmid and two nested PCR assay systems targeting the single copy omp1 gene showed roughly equivalent sensitivity. Importantly, template preparation method and the specific PCR primer system used for screening must be optimised in relation to one another for highest sensitivity.     

Prospective study of toxoplasma reactivation by polymerase chain reaction in allogeneic stem-cell transplant recipients

Toxoplasmosis is a rare but life‐threatening complication of allogeneic stem‐cell transplantation. Polymerase chain reaction (PCR) offers the possibility to make the diagnosis earlier than conventional techniques, and is then expected to improve the prognosis. We undertook a prospective screening using a competitive PCR in blood in 32 stem‐cell transplant recipients. The sampling covered the first 150 days post‐transplant, at days 21, 30, 45, 60, 90, 120, and 150. Twenty‐four patients had anti‐toxoplasma antibodies before transplant. Three of them (12.5%) had transient PCR‐positive samples at 21, 45, and 90 days post‐transplant, respectively. The three PCR‐positive patients were febrile but had no funduscopic examination or cerebral computerised tomography (CT) scan abnormalities. The PCR signal disappeared when the patients were given trimethoprim‐sulfamethoxazole, and no full‐blown toxoplasmosis was observed. Toxoplasma reactivation evidenced using PCR is frequent in seropositive patients not receiving trimethoprim‐sulfamethoxazole during the 1–3 months post‐transplant. Toxoplasma PCR should be included in the diagnostic strategy of fever of unexplained origin in allogeneic stem‐cell transplant recipients. Then, prompt specific therapy can be initiated to avoid development of full‐blown toxoplasmosis ^Note.     

Rapid simultaneous detection of multiple retroviral DNA sequences using the polymerase chain reaction and capillary DNA chromatography

The polymerase chain reaction (PCR) technique is a powerful new tool for amplifying target DNA, thus allowing for sensitive detection of specific nucleic acid sequences. One important potential use of PCR involves screening the donated blood supply for transfusion-transmitted viruses. Realization of this goal has been limited by (1) the requirement for multiple, discrete PCR reactions to amplify and detect target sequences of more than one virus, and (2) the lack of a rapid, nonhazardous means for specific detection of one or more PCR-amplified products. We report the simultaneous amplification of three distinct target sequences without discernable loss in sensitivity toward any single target sequence. We also demonstrate very rapid separation and detection of PCR-amplified viral DNA through the use of automated capillary DNA chromatography. Amplified DNA peaks were initially identified by scanning the capillary effluent at ultraviolet wavelengths, while discrimination of human immunodeficiency virus type 1 and human T-cell leukemic virus type I PCR-amplified DNA was accomplished through use of virus-specific, fluorescently labeled primers and probes. These results indicate progress toward an automated system for screening the blood supply for nucleic acid sequences of multiple pathogens.     

Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction.

The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.     

Multicentre quality control of polymerase chain reaction for detection of HIV DNA.

OBJECTIVE: Seven French laboratories tested the specificity and sensitivity of the polymerase chain reaction (PCR) for the detection of HIV-1 DNA. METHODS: Following its own PCR protocols, each laboratory independently tested blind two panels of 20 coded peripheral blood mononuclear cell samples collected from HIV-1-seropositive individuals and from HIV-1-seronegative individuals at high or low risk of HIV infection. For the first panel, laboratories were free to select type and number of primers; for the second, all were required to use the two primer pairs Pol 3/4 and MMy 9/10' (Nef 1). RESULTS: False-positive and false-negative results were observed in all laboratories (concordance with serology ranged from 40 to 100%). In addition, the number of positive PCR results did not differ significantly between high- and low-risk seronegatives. The use of crude cell lysates in DNA preparation produced the same PCR results as phenol-extracted DNA. Discrepancies between laboratories indicated that factors other than primer pairs contributed strongly to laboratory variability. CONCLUSIONS: Our results emphasize the importance of both positive and negative controls in PCR and demonstrate the value of multicentre PCR quality control.     

A blind study of the polymerase chain reaction for the detection of mycobacterium tuberculosis DNA

Setting: Nine French laboratories routinely involved in mycobacterial work.Objective: To assess the detection of Mycobacterium tuberculosis in experimental samples by polymerase chain reaction (PCR) using the insertion sequence IS6110 as a target for deoxyribonucleic acid (DNA) amplification.Design: Nine laboratories participated in a blind study of the detection of M. tuberculosis by PCR in 20 coded samples containing either a definite number of M. tuberculosis complex (positive samples) or environmental mycobacteria (four samples) or no mycobacteria (five samples).Results: Five laboratories reported false-positive PCR results, with an average rate of 7%. All laboratories except one reported positive PCR results for samples containing 10⁵ cfu/ml or more. M. tuberculosis DNA was detected in two thirds of samples containing 10⁴ and 10³ cfu/ml, and in one third of the samples containing 10² cfu/ml.Conclusion: The results of the study suggest that PCR using IS6110 as a target for DNA amplication is neither very sensitive nor really specific for the detection of M. tuberculosis.     

High detection rate of T-cell receptor beta chain rearrangements in T-cell lymphoproliferations by family specific polymerase chain reaction in combination with the GeneScan technique and DNA sequencing

The distinction between benign polyclonal and malignant monoclonal lymphoid disorders by morphology or immunophenotyping is frequently difficult. Therefore, the demonstration of clonal B-cell or T-cell populations by detecting identically rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is often used to solve this diagnostic problem. Whereas the detection of rearranged Ig genes is well established, TCR gamma (gamma) and beta (beta) gene rearrangements often escape detection with the currently available polymerase chain reaction (PCR) assays. To establish a sensitive, specific, and rapid method for the detection of rearranged TCR-beta genes, we developed a new PCR approach with family-specific Jbeta primers and analyzed the resulting PCR products by high-resolution GeneScan technique. The superior efficiency of this new method was demonstrated by investigating 132 DNA samples extracted from lymph node and skin biopsy specimens (mostly formalin fixed) and blood samples of 62 patients who had a variety of T-cell lymphomas and leukemias. In all but 1 of the tumor samples (98.4%) a clonal amplificate was detectable after TCR-beta PCR and the same clonal T-cell population was also found in 15 of 18 (83%) of the regional lymph nodes and in 7 of 11 (64%) of the peripheral blood samples. Direct comparison of these results with those obtained currently by the most widely applied TCR-gamma PCR revealed an approximate 20% lower detection rate in the same set of samples than with the TCR-beta PCR method. These results indicate that the new TCR-beta PCR is most suitable for a rapid and reliable detection of clonal T-cell populations. (Blood. 2000;96:640-646)