The polymerase chain reaction for Mycoplasma gallisepticum detection

On the basis of the aligned 16S rRNA sequences of Mollicutes, a pair of primers was chosen for the detection of Mycoplasma gallisepticum. When used in the polymerase chain reaction (PCR), the primers detected a specific amplification of all Mg strains tested, yielding an expected 330 bp product. Amplification was not detected when other Mollicutes or E. coli were used as PCR templates. SPF chickens were experimentally inoculated with two strains of M. gallisepticum or Mycoplasma iowae. Tracheal swabs were collected 8, 15, 20 and 28 days after inoculation, and cultured for mycoplasma or tested by PCR. PCR products were detected by hybridization with a digoxigenin-labeled probe and by chemilumines-cence. The results showed that culture was positive for 49/73 swabs while PCR detected 70/72 positive samples. Thus, PCR can provide the basis of a sensitive, specific, rapid and non-radio-active method for detecting M. gallisepticum.     

A T-cell receptor gamma polymerase chain reaction assay using capillary electrophoresis for the diagnosis of cutaneous T-cell lymphomas.

Detection of clonal T-cell receptor gamma rearrangements by polymerase chain reaction (TCRgamma PCR) followed by high-resolution electrophoresis has now become a valuable tool in the diagnosis of cutaneous T-cell lymphoma (CTCL). The identification of clonal TCRgamma PCR products by fluorescent fragment analysis (FFA) on a capillary DNA sequencer is described here and compared with an established hetero-duplex temperature gradient gel electrophoresis (HD-TGGE). Of 55 CTCL derived lesional skin samples, clonality was obtained in 46 samples by FFA (83.6%) and in 45 samples by HD-TGGE (81.8%). Of 35 control skin specimens from various nonmalignant dermatoses, two samples (pityriasis lichenoides chronica) showed clonality by both methods, one sample (chronic dermatitis) only by FFA. The sensitivity of FFA was established using three clonal T-cell lines and peripheral blood mononuclear cells. The detection limit for clonal material was approximately 1% to 2.5% in mixtures of DNA and 1% to 3% in cell dilutions. For cell dilution series, we confirmed a linear correlation between the clonal/polyclonal peak-size ratios and the portion of clonal cells up to about 10%. Thus, the initial ratio between mono-and polyclonal template is correctly displayed by FFA within that concentration range. In conclusion, FFA on capillary DNA sequencer is a well-suited separation technique in TCRgamma PCR-based clonality analysis also exhibiting quantitative properties.     

Use of polymerase chain reaction for detection of Chlamydia trachomatis.

A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved amplified sequences, Southern hybridization, or dot blot analysis. The PCR assay was optimized and, after 40 cycles of amplification with primer set II, demonstrated a sensitivity of 10(-17) g of DNA, which corresponds to the detection of one copy of the plasmid. Because of the high sensitivity, we developed a closed system in which airborne contamination was minimized. Analysis of 228 clinical samples tested by cell culture, IDEIA enzyme immunosorbent assay (Medico-Nobel, Boots-Celltech Ltd., Berkshire, United Kingdom), and PCR showed a sensitivity of 100%, a specificity of 93% when PCR was compared with cell culture, and a corrected specificity of 99% when PCR was compared with cell culture or IDEIA.     

The polymerase chain reaction

The polymerase chain reaction (PCR) is a novel technique that amplifies specific sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealling and extension carried out with the heat stable enzyme, Taq polymerase, leads to exponential increases in the target DNA sequences. The technique has been widely applied in immunology to define HLA polymorphism, T-cell receptor and immunoglobulin diversity, pathogen detection, quantification of lymphokines and lymphoma or leukaemia detection. In addition, it has been very widely applied in all areas of molecular biology and human genetics. Because of its simplicity and sensitivity, PCR represents a major technical development for molecular biologists and will continue to have a great impact on molecular immunology. This review by John Bell describes the basic PCR technique and some variations on it and briefly explores their uses in molecular immunology.     

Rapid polymerase chain reaction assays for Brevibacillus laterosporus detection

The identification of the ubiquitous spore‐forming bacterium Brevibacillus laterosporus, whose interest in pharma, agriculture, and other industrial sectors is raising, mostly relies on 16S ribosomal RNA gene sequence analysis. However, due to bacterial gene homology, this method appears insufficient for a proper discrimination of this species, so that the availability of other target genes is necessary. Leveraging the morphological and genetic feature uniqueness of B. laterosporus, a sensitive and reliable detection and quantification method based on polymerase chain reaction (PCR) and quantitative PCR assays, respectively, was developed. Targeting a highly conserved spore surface protein‐related gene, B. laterosporus could be easily found in different matrices including soil, food, and insect body. Primer set selectivity was confirmed to be very specific and no false positives or negatives were observed using DNA of different bacterial species as a template. The method developed is also suitable for the rapid identification of newly isolated B. laterosporus strains.     

Detection of antigen receptor gene rearrangements in lymphoproliferative malignancies by fluorescent polymerase chain reaction

Abstract: Monoclonal rearrangements of antigen receptor genes in lymphoproliferative diseases are characterized by the specific sequence and the length of their junctional region, which can be used as markers of the proliferating clone. PCR techniques have greatly simplified routine detection of monoclonal rearrangements. But on the one hand, identification of the sequences requires sequencing methods and on the other hand, sizing of rearrangements by conventional analysis of PCR products on agarose or nondenaturing polyacrylamide gels may be uncertain. We have developed an approach based on amplification of rearranged IGH, TCRG and TCRD locus by fluorescent PCR associated to a computerized analysis of generated PCR products allowing their objective sizing. We tested this method on DNA samples from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia, whose pattern of IGH and TCRG rearrangements had been previously identified by Southern blot techniques. TCRG-PCR assay allowed detection of 100% of rearranged samples. No false-negative results were found but a high rate (60%) of Southern-negative and PCR-positive samples were identified. TCRD PCR-assay detected VD1JD1 or VD2-D2/3 rearrangements in both acute lymphoblastic leukemia and chronic lymphocytic leukemia samples. IGH PCR assay permitted detection of all known rearranged samples. The sensitivity of these three different PCR assays (1% leukemic cells) was equivalent to that of other published PCR protocols. These results show the validity and reliability of the fluorescent PCR method for routine detection of IGH, TCRG and TCRD rearrangements. Sizing of PCR products by computerized analysis was also validated. It provides additional information on rearrangement patterns in lymphoproliferative diseases, as clonal rearrangements can be recognized by their size. This can be of great interest in various circumstances, particularly for detection and follow-up of oligoclonality.     

聚合酶链反应技术检测非霍奇金淋巴瘤抗原受体基因重排的局限性

病理形态学和免疫组织化学有效标记,结合分子病理学的检测是当前临床病理诊断的发展趋势。近年来这种三结合的诊断模式已在淋巴瘤的病理诊断中得到应用。各类淋巴瘤的病理形态学特征是病理诊断的基石,免疫组织化学有助于正确的诊断及分类,仅靠这二者,恶性淋巴瘤的误诊率仍较高,采用分子病理学技术检测病理标本中淋巴瘤特征性的分子标记具有重要的参考价值。其中运用聚合酶链反应（PCR）技术检测IgH／TCR基因的重排是目前常用的淋巴瘤的分子病理学的方法之一。     

Combined polymerase chain reaction approach for clonality detection in lymphoid neoplasms.

The present study analyzes the efficiency of a combination of four immunoglobulin heavy chain (IgH) gene polymerase chain reaction (PCR) primer systems and a multiplex T-cell receptor gamma chain (TRG) gene PCR for detection of clonality in 409 samples (234 paraffin sections, 175 bone marrow aspirates) of different lymphomas. Using the four IgH PCR systems together, clonality was detected in all samples of B-cell chronic lymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and Burkitt-like B-cell lymphomas. Clonality was detected in all bone marrow aspirates with lymphoplasmacytoid immunocytoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and unclassifiable low-grade B-cell lymphomas. The combined IgH gene PCR approach allowed clonality detection in 78.2% of myelomas, 75% of Burkitt lymphomas, 74.4% of diffuse large B-cell lymphomas, 68.7% of follicular center lymphomas, 50% of posttransplant lymphomas, 28.6% of anaplastic large cell lymphomas, 29% of T-cell lymphomas, and 18.8% of Hodgkin diseases. The combination of the four IgH gene primer systems with the multiplex TRG gene PCR allowed detection of clonality in 84.2% of B-cell neoplasms, 92.1% of T-cell non-Hodgkin lymphomas, and 18.8% of Hodgkin diseases, which was much more efficient than single PCR protocols.     

Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

The nested polymerase chain reaction technique was compared with the conventional smear and culture methods for detection of Mycobacterium tuberculosis. The nested polymerase chain reaction used in this study showed excellent specificity, sensitivity, and agreement with the conventional methods for 417 clinical samples, indicating a contribution to the rapid diagnosis of mycobacterial infectious diseases.     

聚合酶链反应检测沙眼衣原体的临床应用

本文应用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）技术对１０５例非淋菌性尿道炎患者进行沙眼衣原体检测，捡出阳性者１２例，阳性率１１．４％。我们认为ＰＣＲ法检测沙眼衣原体具有快速简便、高度敏感性和特异性等优点，值得临床推广应用。     

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.     

Sensitive detection of Helicobacter pylori by using polymerase chain reaction.

A polymerase chain reaction (PCR) for the specific detection of Helicobacter pylori was developed with a single primer pair derived from the nucleotide sequence of the urease A gene of H. pylori. We achieved specific amplification of a 411-bp DNA fragment in H. pylori. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis and contained conserved single HinfI and AluI restriction sites. This fragment was amplified in all 50 strains of H. pylori tested, but it was not detected in other bacterial species, showing the PCR assay to be 100% specific. PCR DNA amplification was able to detect as few as 10 H. pylori cells. PCR detected H. pylori in 15 of 23 clinical human gastric biopsy samples, whereas culturing and microscopy detected H. pylori in only 7 of the samples found to be positive by PCR. Additional primer pairs based on the urease genes enabled the detection of H. pylori in paraffin-embedded human gastric biopsy samples. The detection of H. pylori by PCR will enable both retrospective and prospective analyses of clinical samples, elucidating the role of this organism in gastroduodenal disease.     

The use of the polymerase chain reaction for the detection of human papillomavirus type 13.

Human papillomavirus type 13 (HPV-13) is associated with oral focal epithelial hyperplasia (FEH). The purpose of this study was to establish conditions for the application of polymerase chain reaction (PCR) to the specific detection and amplification of HPV-13 DNA. To design primers for HPV-13 a part of the HPV-13 genome was sequenced first: the smallest BamHI fragment (597 bp) of HPV-13 was subcloned and sequenced. The sequence was found to be part of a large open reading frame and had significant homology with the L1 gene of other HPVs. HPV-13 specific primers were designed to amplify a 240 bp fragment from the L1 gene by PCR. Conditions for PCR were standardized for this set of primers.