Competitor internal standards for quantitative detection of mycoplasma DNA

Abstract Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumonias . To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.     

A PCR-based marker for selection of starch and potential noodle quality in wheat

A strong association between the absence of the granule-bound starch synthase (GBSS) protein for the 4A chromosome of wheat and Japanese Udon noodle quality has been previously described. The aim of this study was to identify a molecular marker linked to the GBSS 4A locus which could be used to identify wheat with the desired texture for Udon noodles. PCR primers were designed to target this gene which gave a 440 bp PCR band, corresponding to the presence or absence of the 4A GBSS gene. Of the 268 genotypes screened with these primers, 267 were correctly identified using the PCR primers. The remaining genotype was shown to be heterogeneous for the marker. The PCR marker test developed has advantages over existing methods used to screen for Udon noodle starch quality as it enables high throughput, accurate tests to be carried out on leaves of young seedlings or mature seed and identify breeding lines that are heterogeneous for the 4A allele which will allow for reselections. Application of this PCR test will speed up selection for Udon noodle quality genotypes and reduce breeding costs for production of noodle wheat varieties. Abbreviations: CTAB, cetyltrimethlammonium bromide; FSV, flour swelling volume; GBSS, granule-bound starch synthase; IEF, isoelectric focusing; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.     

转基因作物及加工品检测技术概述

随着公众对转基因产品关注度的提高和转基因标识制度的建立,快速、准确、灵敏地检测出转基因成分是客观发展的必然要求,建立高效、高通量的转基因检测技术已成为国内外的热点课题。我们简要介绍了转基因检测技术的概念、原理和研究进展,比较了各种转基因检测技术的优缺点,对转基因检测技术的发展方向进行了总结和展望。     

Evaluation of Bacteroides markers for the detection of human faecal pollution

Aims: This paper reports on the results of a study aimed at evaluating the specificity and sensitivity of human‐specific HF183 and HF134 Bacteroides markers in various host groups and their utility to detect human faecal pollution in storm water samples collected from nonsewered catchments in Southeast Queensland, Australia. Methods and Results: The specificity and sensitivity of the HF183 and HF134 Bacteroides markers was evaluated by testing 207 faecal samples from 13 host groups, including 52 samples from human sources (via sewage and septic tanks). Polymerase chain reaction analysis of these samples revealed the presence/absence of HF183 and HF134 across these host groups, demonstrating their suitability for distinguishing between human and animal faecal pollution. The HF183 marker was found to be more reliable than that of HF134, which was also found in dogs. Conclusions: Based on our data, it appears that the HF183 marker is specific to sewage and is a reliable marker for detecting human faecal pollution, while the use of HF134 marker alone may not be sufficient enough to provide the evidence of human faecal pollution. Significance and Impact of the Study: This is the first study in Australia that rigorously evaluated the specificity and sensitivity of Bacteroides markers. Based on our findings, we suggest that the HF183 marker could reliably be used to detect the sources of human faecal pollution in Southeast Queensland region.     

Comparison of different primers for rapid detection of Vibrio parahaemolyticus using the polymerase chain reaction

AIMS: The aim of this study was to compare different primers for rapid and effective detection of Vibrio parahaemolyticus by polymerase chain reaction (PCR). METHODS AND RESULTS: A total of four pairs of primers, three previously published and one based on a newly developed V. parahaemolyticus metalloprotease (vpm) gene, have been assayed for PCR detection of V. parahaemolyticus. They have been tested for specificity and sensitivity on a total of 101 strains including reference and environment isolates belonging to V. parahaemolyticus and other species in Vibrio. Of the four sets of primers tested, the one designed on the basis of the metalloprotease gene (675 bp) gave optimal results with bacterial strains examined as they only amplified the specific fragment in strains that had been genetically and biochemically assessed as V. parahaemolyticus and the limit of detection was 4 pg of purified target DNA. CONCLUSIONS: The primers designed on the metalloprotease gene gave optimal results for specific, sensitive and rapid detection of V. parahaemolyticus by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplification with the optimal primer set VPM1/VPM2 could facilitate the rapid diagnosis and surveillance of potentially pathogenic strains of V. parahaemolyticus and reduce food-borne illness.     

Development of a combined selective enrichment method and polymerase chain reaction (PCR) assay for sensitive detection of Salmonella in food samples

PCR assays were formatted using primer pairs homologous to phoE and invA genes. The amplification conditions were optimized with pure cultures and reactions were carried out to define selectivity, specificity and sensitivity of both primer pairs. The performance of the invA primer pair was better than that of the phoE pair, making the specific detection of Salmonella serovars and strains isolated from different food samples possible. Using the invA primer pair, the combined selective enrichment method with the polymerase chain reaction assay was established and used to detect Salmonella from artificially multi-contaminated food samples. The complete procedure detected as few as three cells of Salmonella (3 c.f.u.) from milk and meat samples.     

In vitro culture of Gypsophila paniculata L. and random amplified polymorphic DNA analysis of the propagated plants

A protocol is established for regeneration of the economically important cut flower plant, Gypsophila paniculata L., using shoot tips explants. Multiple shoots were obtained on Murashige and Skoog medium fortified with 0.5 mg dm⁻³ each of α-naphthaleneacetic acid and 6-benzyladenine. Addition of 10 g dm⁻³ agar promoted shoot proliferation and reduced the degree of shoot vitrification. Transfer to 3 mg dm⁻³ indole⁻³-butyric acid containing medium produced optimum root initiation and development. The produced plants as well as intact plants were subjected to the random amplified polymorphic DNA (RAPD) analysis. Using 9 primers, the total number of amplification products generated by polymerase chain reaction was 142 bands (15.7 bands per primer), of which 7.74 % showed polymorphism. The analysis of bands recorded, showed 92.25 % similarity. The results indicated that very low variation at the DNA level occurred during in vitro culture of Gypsophila.     

smcL as a novel diagnostic marker for quantitative detection of Listeria ivanovii in biological samples

Aims: To develop a novel molecular tool for the quantitative detection of the ruminant pathogen Listeria ivanovii in different biological matrices. Methods and Results: A real‐time PCR (RTi‐PCR) for the quantitative and species‐specific identification of L. ivanovii was designed to target the region of the smcL gene. The assay includes an internal amplification control (IAC) to avoid false‐negative results. The smcL‐IAC RTi‐PCR assay was 100% selective and allowed the detection of as little as one genome equivalent in 45% of reactions. The quantification accuracy was excellent, as demonstrated by its high linearity (R² > 0·9989) and PCR efficiency (E > 0·984) over a 6‐log dynamic range, down to 10 genome equivalents. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk, blood and amniotic fluid. The smcL‐IAC RTi‐PCR assay allowed the detection of as few as 50 colony forming unit numbers (CFUs) per 25 ml of raw milk, 43 CFUs per 1 ml of blood or 50 CFUs per 1 ml of amniotic fluid. Conclusions: This method can be an adequate alternative for the identification of L. ivanovii and for complete diagnosis of animal and human listeriosis. Significance and Impact of the Study: We present an alternative for the detection of another pathogenic member of Listeria genus, which can help to distinguish from Listeria monocytogenes and therefore facilitates the establishment of preventive and prophylactic measures in food and farm environments.     

The use of an arbitrarily primed PCR product for the specific detection of Brucella

A 1.3 kb Brucella-specific DNA fragment produced through the use of arbitrarily primed polymerase chain reaction (AP-PCR) was tested for its specificity by DNA–DNA hybridization to Brucella and non-Brucella bacteria. The digoxigenin (DIG)-labelled 1.3 kb DNA fragment hybridized with Brucella abortus and Brucella melitensis but did not hybridize with other non-Brucella bacteria tested. The sensitivity of the reaction was determined; as little as 150 fg DNA or 30 Brucella cells could be detected. The specificity and sensitivity of the 1.3 kb DNA fragment combined with the simplicity and speed of the technique suggest the potential of this fragment as a DNA probe for the quick and reliable detection of Brucella organisms.     

Detection of mitochondrial DNA deletions by a screening procedure using the Polymerase Chain Reaction

We describe an accurate procedure for a rapid diagnosis of heteroplasmic mtDNA deletions based on the polymerase chain reaction (PCR). For a selective amplification of deleted mtDNA across the breakpoints of the deletion, we used seven combinations of primers surrounding the most common deleted region between the two origins of mtDNA replication. This procedure was performed on muscle biopsies of twenty patients harboring a single mtDNA deletion and one patient with multiple mtDNA deletions. The results were compared with Southern-blotting analysis. We conclude that this PCR procedure is a sensitive and convenient screening method for the detection of mtDNA deletions. (Mol Cell Biochem 174: 221–225, 1997)     

Consistency in the host specificity and host sensitivity of the Bacteroides HF183 marker for sewage pollution tracking

Aims: The host specificity (H‐SPF) and host sensitivity (H‐SNV) values of the sewage‐associated HF183 Bacteroides marker in the current study were compared with the previously published studies in South East Queensland (SEQ), Australia, by testing a large number of wastewater and faecal DNA samples (n = 293) from 11 target and nontarget host groups. This was carried out to obtain information on the consistency in the H‐SPF and H‐SNV values of the HF183 marker for sewage pollution tracking in SEQ. Methods and Results: Polymerase chain reaction (PCR) analysis was used to determine the presence/absence of the HF183 marker in wastewater and faecal DNA samples. Among the human composite wastewater (n = 59) from sewage treatment plants and individual human (n = 20) faecal DNA samples tested, 75 (95%) were PCR positive for the HF183 marker. The overall H‐SNV of this marker in target host group was 0·95 (maximum of 1·00). Among the 214 nontarget animal faecal DNA samples tested, 201 (94%) samples were negative for the HF183 marker. Six chicken, five dog and two bird faecal DNA samples, however, were positive for the marker. The overall H‐SPF of the HF183 marker to differentiate between target and nontarget faecal DNA samples was 0·94 (maximum of 1·00). Conclusions: The H‐SNV (0·95) and H‐SPF (0·94) values obtained in this study was slightly lower than previous studies (H‐SNV value of 1·00 in 2007 and 1·00 in 2009; H‐SPF value of 1·00 in 2007 and 0·99 in 2009). Nonetheless, the overall high H‐SNV (0·98) and H‐SPF (0·97) values of the HF183 marker over the past 4 years (i.e. 2007–2011) suggest that the HF183 marker can be reliably used for the detection of sewage pollution in environmental waters in SEQ. Significance and Impact of the Study: In the current study, the HF183 marker was detected in small number nontarget animal faecal samples. Care should be taken to interpret results obtained from catchments or waterways that might be potentially contaminated with dog faecal matter or poultry litter.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

Microsatellite marker development,mapping and applications in rice genetics and breeding

Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily and economically assayed by the polymerase chain reaction (PCR). Results from screening a rice genomic library suggest that there are an estimated 5700-10 000 microsatellites in rice, with the relative frequency of different repeats decreasing with increasing size of the motif. A map consisting of 120 microsatellite markers demonstrates that they are well distributed throughout the 12 chromosomes of rice. Five multiple copy primer sequences have been identified that could be mapped to independent chromosomal locations. The current level of genome coverage provided by these simple sequence length polymorphisms (SSLPs) in rice is sufficient to be useful for genotype identification, gene and quantitative trait locus (QTL) analysis, screening of large insert libraries, and marker-assisted selection in breeding. Studies of allelic diversity have documented up to 25 alleles at a single locus in cultivated rice germplasm and provide evidence that amplification in wild relatives of Oryza sativa is generally reliable. The availability of increasing numbers of mapped SSLP markers can be expected to complement existing RFLP and AFLP maps, increasing the power and resolution of genome analysis in rice.     

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.     

Evaluation and applicability of a purification method coupled with nested PCR for the detection of Toxoplasma oocysts in water

AIMS: To describe the development, evaluation and applicability of a complete method for the detection of Toxoplasma gondii in water. METHODS AND RESULTS: The method incorporated concentration of water samples by Al(2)(SO(4))(3)-flocculation, purification by discontinuous sucrose gradients and detection of toxoplasmic DNA by 18S-rRNA nested PCR. Tap water replicates and natural water samples were seeded with defined numbers of Toxoplasma oocysts and processed for evaluation studies. When applied to environmental samples, the method gave highest detection sensitivities of 100 oocysts in river water and 10 oocysts in well- and sea water. The method was finally applied in 60 water samples of different quality and origin collected over a 14-month period. Toxoplasmic DNA was detected in four samples. CONCLUSIONS: The method offers an alternative towards improving current methods that can be used for the detection of Toxoplasma oocysts in environmental water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method in its current form will be helpful for assessment of Toxoplasma contamination in water resources, particularly after outbreak events.