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Newly Published PCR Papers - Updated Daily
PCR-based detection and eradication of mycoplasmal infections from various ma...
PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience.
Cytotechnology. 2010 Feb 6;
Authors: Molla Kazemiha V, Shokrgozar MA, Arabestani MR, Shojaei Moghadam M, Azari S, Maleki S, Amanzadeh A, Jeddi Tehrani M, Shokri F
A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.
PMID: 20135349 [PubMed - as supplied by publisher]
Real-Time PCR Expression Profiling of MMPs and TIMPs.
Real-Time PCR Expression Profiling of MMPs and TIMPs.
Methods Mol Biol. 2010;622:159-73
Authors: Ennington CJ, Edwards DR
Quantitative reverse transcriptase polymerase chain reaction enables the accurate quantification of gene expression in cultured cells or small tissue samples. In this chapter, we describe the use of Taqman((R)) technology to measure expression of matrix metalloproteinases and related genes.
PMID: 20135281 [PubMed - in process]
A Real-time PCR assay for the quantitative detection of Ralstonia solanacearu...
A Real-time PCR assay for the quantitative detection of Ralstonia solanacearum in the horticultural soil and plant tissues.
J Microbiol Biotechnol. 2010 Jan;20(1):193-201
Authors: Chen Y, Zhang WZ, Liu X, Ma ZH, Li B, Allen C, Guo JH
A specific and rapid real-time PCR assay for detecting Ralstonia solanacearum in the horticultural soil and plant tissues was developed in this study. The specific primers RSF/RSR were designed based on the upstream region of UDP-3-O-acyl-GlcNAc deacetylase gene from R. solanacearum, and a PCR product of 159 bp was amplified specifically from 28 strains of R. solanacearum, which represent all genetically diverse AluI types and all 6 biovars, but not from any other nontarget species. The detection limit of 102 CFU/g tomato stem and horticultural soil was achieved in this real-time PCR assay. The high sensitivity and specificity observed with filed samples as well as with artificially infected samples suggested that this method might be a useful tool for detection and quantification of R. solanacearum in precise forecast and diagnosis.
PMID: 20134252 [PubMed - in process]
Real-time PCR Analysis of metabolic pathway of PHB in Acidiphilium cryptum DX...
Real-time PCR Analysis of metabolic pathway of PHB in Acidiphilium cryptum DX1-1.
J Microbiol Biotechnol. 2010 Jan;20(1):71-7
Authors: Xu AL, Xia JL, Liu KK, Li L, Yang Y, Nie ZY, Qiu GZ
The time, yield and related genes expression of PHB accumulation of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios 1.2, 2.4, 7.5, and 24. The results of time and yield of PHB accumulation show that the initial C/N ratio 2.4 was optimum for strain DX1-1 to accumulate PHB, both higher and lower initial C/N ratios did not favor that process. Based on the genome of Acidiphilium cryptum JF-5, 13 PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expression of the 13 functional genes, in different C/N ratios as cited above, was then studied by Real-time PCR. The results show that all the 13 genes were most upregulated when initial C/N ratio was 2.4, and among which the gene Acry_3030 encoding poly-beta-hydroxybutyrate polymerase and Acry_0626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which prove that they play the most important role for PHB accumulation and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis shows that the genes related to PHB accumulation are regulated by different promoters and that the motif had weak similarity to the model promoters, suggesting that PHB- metabolism in Acidiphilium cryptum may be mediated by a different mechanism.
PMID: 20134235 [PubMed - in process]
Analysis of peripheral immune activation in schizophrenia using quantitative ...
Analysis of peripheral immune activation in schizophrenia using quantitative reverse-transcription polymerase chain reaction (RT-PCR).
Psychiatry Res. 2010 Feb 2;
Authors: Freudenreich O, Brockman MA, Henderson DC, Evins AE, Fan X, Walsh JP, Goff DC
Immune system abnormalities in schizophrenia include a shift from a Type 1 (cellular) to a Type 2 (humoral) immune response. To characterize the activation status of the immune system in schizophrenia, we examined the pattern of gene expression in peripheral blood cells for three Th1 cytokines (interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2)), and one Th2 cytokine (interleukin-10 (IL-10)). In a cross-sectional study, we used quantitative reverse-transcription polymerase chain reaction (RT-PCR) to compare the mRNA levels of IFN-gamma, TNF-alpha, IL-2, and IL-10 in peripheral blood mononuclear cells (PBMCs) between 15 schizophrenia patients and 15 matched healthy controls. Expression of IFN-gamma and TNF-alpha was significantly reduced in patients with schizophrenia compared with normal controls. No differences in IL-2 and IL-10 gene expression were observed. These results are consistent with impaired Type 1 cellular immunity in schizophrenia. While the data illustrate the potential utility of mRNA-based approaches for the identification and analysis of immune biomarkers for neuropsychiatric disorders, correlation of gene expression with direct measures of cytokine concentrations is required.
PMID: 20132993 [PubMed - as supplied by publisher]
Comparison between Quantitative Real-Time Reverse Transcription PCR Results f...
Comparison between Quantitative Real-Time Reverse Transcription PCR Results for Norovirus in Oysters and Self-Reported Gastroenteric Illness in Restaurant Customers.
J Food Prot. 2010 Feb;73(2):305-11
Authors: Lowther JA, Avant JM, Gizynski K, Rangdale RE, Lees DN
Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This study attempts to compare norovirus RNA detection in Pacific oysters (Crassostrea gigas) by quantitative real-time reverse transcription PCR (RT-PCR) and human health risk. Self-reported customer complaints of illness in a restaurant setting (screened for credible norovirus symptoms) were compared with presence and levels of norovirus as determined by real-time RT-PCR for the batch of oysters consumed. No illness was reported for batches consistently negative for norovirus by real-time RT-PCR. However, norovirus was detected in some batches for which no illness was reported. Overall presence or absence of norovirus showed a significant association with illness complaints. In addition, the batch with the highest norovirus RNA levels also resulted in the highest rate of reported illness, suggesting a linkage between virus RNA levels and health risks. This study suggests that detection of high levels of norovirus RNA in oysters is indicative of a significantly elevated health risk. However, illness may not necessarily be reported after detection of norovirus RNA at low levels.
PMID: 20132676 [PubMed - in process]
Rapid Detection and Differentiation of Campylobacter jejuni, Campylobacter co...
Rapid Detection and Differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in Food, Using Multiplex Real-Time PCR.
J Food Prot. 2010 Feb;73(2):241-50
Authors: Mayr AM, Lick S, Bauer J, Thärigen D, Busch U, Huber I
A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.
PMID: 20132668 [PubMed - in process]
A quantitative PCR assay for rapid detection of Shigella species in fresh pro...
A quantitative PCR assay for rapid detection of Shigella species in fresh produce.
J Food Prot. 2010 Feb;73(2):221-33
Authors: Lin WS, Cheng CM, Van KT
A quantitative PCR (qPCR) assay with two primers and a TaqMan probe targeting conserved regions of the specific ipaH gene of Shigella species and enteroinvasive Escherichia coli (EIEC) were developed. This qPCR assay was used to identify 206 Shigella strains (including four Shigella species with all serotypes and two provisional Shigella species), 3 EIEC strains, and 113 non-Shigella strains with 100% accuracy. Pure cultures of six Shigella reference strains were used to derive standard curves to determine the detection limit and efficiency of the qPCR method. The ipaH qPCR assay had an equally low detection limit (0.12 to 0.74 CFU per PCR) for the four Shigella species tested. The average qPCR efficiency was 99.29% (95.36 to 103.92%). The detection limit of the qPCR assay tested on 15 varieties of inoculated fresh produce ranged from 0.4 to 16 CFU/100 ml of buffer rinse. This qPCR assay took the variation of wild-type nucleotides into consideration and was used successfully to screen fresh produce. This highly sensitive qPCR assay can be completed within 24 h and has potential use as a screening tool for all four Shigella species and EIEC in food samples.
PMID: 20132666 [PubMed - in process]
Enterococcus species distribution among human and animal hosts using multiple...
Enterococcus species distribution among human and animal hosts using multiplex PCR.
J Appl Microbiol. 2010 Jan 11;
Authors: Layton BA, Walters SP, Lam LH, Boehm AB
Abstract Aims: This study evaluated the use of Enterococcus species differentiation as a tool for microbial source tracking (MST) in recreational waters. Methods and Results: Avian, mammalian and human faecal samples were screened for the occurrence of Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae and Enterococcus saccharolyticus using multiplex PCR. Host-specific patterns of Enterococcus species presence were observed only when data for multiple Enterococcus species were considered in aggregate. Conclusions: The results suggest that no single Enterococcus species is a reliable indicator of the host faecal source. However, Enterococcus species composite 'fingerprints' may offer auxiliary evidence for bacterial source identification. Significance and Impact of Study: This study presents novel information on the enterococci species assemblages present in avian and mammalian hosts proximate to the nearshore ocean. These data will aid the development of appropriate MST strategies, and the approach used in this study could potentially assist in the identification of faecal pollution sources.
PMID: 20132375 [PubMed - as supplied by publisher]
Assessment of RNA amplification by multiplex RT-PCR and IgM detection by indi...
Assessment of RNA amplification by multiplex RT-PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella.
APMIS. 2010 Mar;118(3):203-9
Authors: Sanz JC, Mosquera M, Ramos B, Ramírez R, DE Ory F, Echevarria JE
Sanz JC, Mosquera M, Ramos B, Ramírez R, de Ory F, Echevarria JE. Assessment of RNA amplification by multiplex RT-PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella. APMIS 2010; 118: 203-9. The aim of the study was to compare RNA amplification using multiplex RT-PCR and IgM detection by means of indirect and capture ELISAs for the diagnosis of measles and rubella. A total of 229 cases of maculopapular rash with serum and throat swab samples were included. Specific serological IgM to measles and rubella was determined by Enzygnost((R)) (Siemens) and Platelia(TM) (Bio-Rad). Both viruses were researched using multiplex RT-PCR performed on throat samples. Criteria for inclusion of measles or rubella cases were a positive RT-PCR result for one virus and negative for the other; and/or a positive IgM result for one virus by both ELISAs and negative RT-PCR for the other virus. A total of 74 cases were classified as measles and 54 as rubella. In measles, sensitivity and specificity were 93.2% and 100% for RT-PCR, 97.3% and 98.1% for Enzygnost((R)), and 90.5% and 95.5% for Platelia(TM). For rubella, these values were 42.6% and 100% for RT-PCR, 100% and 97.1% for Enzygnost((R)), and 94.4% and 98.3% for Platelia(TM). Enzygnost((R)) and Platelia(TM) are useful techniques for detecting IgM against measles and rubella. RNA amplification by RT-PCR was both sensitive and specific for the diagnosis of measles; however, for rubella, the sensitivity of this technique must be improved.
PMID: 20132186 [PubMed - in process]