Molecular cloning of human cardiac troponin I using polymerase chain reaction

We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart.     

Diagnosis of bovine freemartinism by the polymerase chain reaction method

Summary. Using sex chromosome specific primers, leucocyte chimaerism in heterosexual bovine female twins was identified by combining polymerase chain reaction (PCR) and restriction enzyme digests.     

Cross-species amplification of glutamine synthetase cDNA by polymerase chain reaction with degenerate primers

We have developed and optimized a consistent polymerase chain reaction (PCR)-based strategy to quickly obtain specific sequence information on novel plant glutamine synthetase (GS, EC 6.3.1.2) cDNAs. Two sets of degenerate primer pairs were designed to discriminate regions conserved in either any kind of GS messenger or exclusively in those for the chloroplastic GS. Novel GS cDNA sequences were successfully amplified from total RNA obtained from 14 different monocotyledonous and dicotyledonous plants. The procedure, coupled with a further restriction analysis, allowed us to uncover the presence of GS cDNA polymorphism, which most likely stems from the different GS gene family members within a single species. Contrary to previously reported strategies in other systems, GS cDNA oligonucleotide primers were designed keeping the degeneracy level to a minimum, together with a high melting temperature. This approach proved to be particularly effective, generating high yields of the expected products without requiring extra nested amplification steps or time-consuming optimization steps for each species GS cDNA amplification. Different clones containing sequence information from either the coding or the 3'-untranslated regions were further sequenced and characterized, confirming the high sequence identity and size uniformity the of GS cDNAs across higher plant species. Therefore, this approach is proposed as a stand-alone procedure to quickly determine the sequence of unknown GS cDNAs, as well as to speed up and complement classical molecular cloning methodologies.     

Detection and identification ofCampylobacter coli andCampylobacter jejuni by two-step polymerase chain reaction

Flagellin gene was used as target sequence to detect and distinguishC. coli andC. jejuni by a “nested PCR” technique. The method shows a high level of sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about epidemiological and pathogenetic implications of each of these two microorganisms.     

Detection of bovine kappa-casein genomic variants by the polymerase chain reaction method

Summary A method is described for identifying variants at the bovine κ-casein locus by combining polymerase chain reaction (PCR) amplification and restriction enzyme digests.     

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.     

Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction

An easy and quick protocol has been developed for DNA analysis via PCR. Single cereal endosperm or small leaf pieces can be separately processed in several PCR reactions. The resultant PCR patterns are equivalents to those obtained with standard DNA extraction protocols using either specific or random primers. Intra-and inter-specific variability can be detected. This method allows the analysis of a large number of individuals in early stages prior to the plant sowing.     

数字聚合酶链反应及其在感染性疾病中的应用

数字聚合酶链反应(polymerase chain reaction,PCR)采用与定量PCR相同的荧光化学原理和不同的数学原理来实现对靶标核酸序列的绝对定量,其摒弃了对外部参照的依赖,同时具有更高的数据精密度,提高了重复性和再现性。数字PCR的应用涵盖生命科学众多领域,特别是在医学检验领域,其对疾病相关核酸分子标记的准确分析,为疾病的早期诊断、进展监测、疗效评估提供了动态量化指标。数字PCR的出现将推动基于核酸扩增技术的分子生物学检测迈入精准定量阶段。本文就数字PCR尤其是微滴式数字PCR在感染性疾病中的应用进展及前沿进行综述。     

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction

Abstract Candida species are opportunistic human pathogens capable of causing a variety of clinical diseases. Rapid and precise identification of Candida to species level is essential for effective treatment and management strategies. Conventional diagnosis of candidiasis is sometimes slow and variable. By using a random primer 5'-ACGGGCCAGT-3' in arbitrarily primed polymerase chain reaction, seven common Candida species (i.e. C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. lipolytica, C. tropicalis and C. (Torulopsis) glabrata ) produced characteristic DNA band patterns, which enabled rapid determination to species level among them. Further analysis of the nucleotide sequences of Candida specific fragments should improve our understanding of the molecular structures and possible functions of the gene regions involved.     

Prevalence of Theileria sergenti infection in Korean native cattle by polymerase chain reaction

This study was performed to investigate the prevalence of theileriosis and to compare the prevalence of this disease in Korean native cattle reared under different environmental conditions, namely, in a grazing area and a non-grazing area by polymerase chain reaction. Three hundred and one Korean native cattle (276 cows and 25 bulls) that had not received prior treatment or been vaccinated to prevent theileriosis were examined by PCR for Theileria sergenti infection from 2001 to 2002. In our study, the parasitemia range in T. sergenti-positive cattle by microscopy were from 0.1 to 3% (mean 0.8%). In terms of mean prevalence, 204 of the 301 Korean native cattle (67.8%) were positive reaction by PCR. Our results also revealed that the infection rate among cows (70.3%) was significantly higher than that among bulls (40.0%) (p < 0.01). T. sergenti infection among the over 3 year-old-group (75%) had a significant higher prevalence than that among the less than 3 year-old-group (61.8%) (p < 0.05). Our data also showed that grazing areas (76.1%) had the significant higher prevalence than non-grazing areas (51%) (p < 0.001). In conclusion, this study demonstrates that the prevalence of T. sergenti infection is high and that its prevalence in grazing cattle is higher than that in non-grazing cattle. Therefore, life-long treatment and the development of an optimal vaccine are needed to reduce the numbers of bovine theileriosis in both grazing and non-grazing areas.     

Rapid detection of Haemophilus influenzae by hel gene polymerase chain reaction

AIMS: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. METHODS AND RESULTS: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65.9%) of 91 samples in contrast to 62 (68.12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. CONCLUSIONS: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods.     

Significance of high-risk human papillomavirus detection by polymerase chain reaction in primary cervical cancer screening

The purposes of this study were to evaluate the incidence of high-risk human papillomavirus (HPV) infection by polymerase chain reaction (PCR) and to assess its diagnostic usefulness in primary cervical screening. PCR testing for HPV type 16, 18, 31 and 33 was performed on 1305 specimens obtained during routine cervical cancer screening. We analysed the concurrent cervical smears and biopsy, and correlated them with the HPV infection status. We also evaluated histologically-proven cases with ASCUS smears according to HPV infection. HPV DNA was identified in eight (0.7%) of 1144 cytologically normal patients; nine (10.5%) of 86 ASCUS; seven (25.0%) of 28 LSIL; 26 (78.8%) of 33 HSIL; and in all of three squamous cell carcinomas (SCC). HPV positivity was significantly associated with cytohistological diagnosis for HSIL of more. In addition, HPV-positive ASCUS cases were found to be associated with histological abnormality rather than HPV-negative. The results indicate that high-risk HPV testing by PCR could be a useful adjunct tool for Pap smear in primary cervical screening. The combination of Pap smear and high-risk HPV testing by PCR might reduce unnecessary colposcopy-guided biopsy of women with cytological diagnosis of ASCUS.     

Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction targeting the actA gene

AIMS: The aim of this study was to develop a 5'-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes. METHODS AND RESULTS: Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 10(4) cfu ml(-1) after 35 cycles and 10(2) cfu ml(-1) after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 10(2) to 10(9) cfu ml(-1) for three L. monocytogenes strains in real-time PCR with 45 cycles. CONCLUSIONS: The developed 5'-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.     

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.