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Results for “Reverse Transcriptase Polymerase Chain Reaction”

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At least 19 recordsLinked to original sources
PubMed1998

Quantification of poliovirus in seawater and sewage by competitive reverse transcriptase--polymerase chain reaction.

Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5'-carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.

Environmental Microbiology↗
PubMed1993

Early detection of de novo hepatitis C infection in patients after liver transplantation by reverse transcriptase polymerase chain reaction.

BACKGROUND: Reverse transcriptase polymerase chain reaction (RT-PCR) can detect the viral genome and show hepatitis C recurrence in patients who undergo transplantation for chronic hepatitis C viral (HCV) infection. We investigated the utility of an RT-PCR-based HCV assay for early detection of viral RNA in de novo HCV infection after liver transplantation. METHODS: Pretransplantation antibodies and explanation HCV viral RNA status were obtained from 117 patients. Follow-up liver biopsy specimens were examined for evidence of hepatitis activity. Plasma samples during the period of time of the biopsy were assayed for HCV antibody and viral RNA. RNA was extracted from samples and reverse transcribed to cDNA. cDNA was amplified by PCR, and products were detected by liquid hybridization. RESULTS: Clinical hepatitis developed in seventeen of 117 patients who, before transplantation, were HCV antibody negative and explant viral RNA negative. Ten patients were plasma PCR negative and had known non-hepatitis C causes for the biopsy findings. Of the remaining seven patients, five (70%) were plasma RT-PCR positive before seroconversion in matched plasma samples. CONCLUSIONS: In liver transplant patients, the incidence of de novo clinical hepatitis is low, and HCV viral RNA in de novo clinical hepatitis C infection can be detected in the absence of HCV antibodies.

Base Sequence↗
PubMed1997

Detection of pancreatic and gastric cancer cells in peripheral and portal blood by amplification of keratin 19 mRNA with reverse transcriptase-polymerase chain reaction.

Reverse transcriptase-polymerase chain reaction (RT-PCR) targeted at keratin 19 mRNA was applied to detect circulating cancer cells in the peripheral and portal blood of pancreatic and gastric cancer patients. Keratin 19 mRNA expression was studied by RT-PCR in cancer tissues (12 pancreatic and 15 gastric cancers) and in peripheral and/or portal blood samples from patients with pancreatic cancer (stage I, n = 5; stage II, n = 1; stage III, n = 15; stage IV, n = 19), gastric cancer (stage la,b, n = 28; stage II, n = 9; stage IIIa,b, n = 5; stage IVa,b, n = 7) and benign pancreatic diseases (n = 7). Peripheral blood samples from 50 healthy volunteers served as controls. RT-PCR was conducted in duplicate in each sample, and only samples showing keratin 19 transcript in both determinations were considered as being positive. All the pancreatic and gastric cancers, but none of the control blood samples, were found to be positive. Dilution study using pancreatic cancer cells serially mixed against peripheral blood showed that detection sensitivity was more than one cancer cell in 10(6) peripheral blood mononuclear cells. In pancreatic cancer patients, RT-PCR analysis of the portal blood samples gave positive results in one stage III and one stage IV patient, and that of peripheral blood samples gave positive results in 2 stage IV patients. No positive results were obtained in any of the blood samples from gastric cancer patients. Our results indicate that incidence of circulating cancer cells is unexpectedly very low even in advanced pancreatic and gastric cancer patients.

Humans↗
PubMed1995

Mathematical considerations of competitive polymerase chain reaction.

Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1-5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals.

Animals↗
PubMed1999

Detection of circulating prostate specific antigen expressing prostatic cells in the bone marrow of radical prostatectomy patients by sensitive reverse transcriptase polymerase chain reaction.

PURPOSE: The reverse transcriptase polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) expressing cells in the blood circulation has been under intense investigation since 1992. Although it has been suggested that this technology could be used as molecular staging for occult prostatic hematogenous metastases, we have been unable to confirm RT-PCR PSA positivity of peripheral blood to predict stage or recurrence in radical prostatectomy cases. We performed bone marrow RT-PCR PSA assay on a large cohort of radical prostatectomy cases and evaluate the use of this assay in improving prostate cancer staging and detecting early recurrence. MATERIALS AND METHODS: Unilateral anterior iliac crest bone marrow aspirates were performed on 116 patients immediately before radical prostatectomy between February 1995 and September 1997. Radical prostatectomy specimens were processed as whole mounts. A sensitive nested RT-PCR assay with specific primers derived from the PSA sequence was used, which enabled us to detect PSA expressing LNCaP prostate cancer cells at the sensitivity of 1 cancer cell per 10 million lymphocytes (1/10(7)). A minimum of 3 RT-PCR PSA reactions were performed on all patients and at least 2 positive tests were required to define positivity. Patients were followed for PSA recurrence (mean followup 14.7 months). RESULTS: PSA expressing cells were detected in bone marrow of 51 of 116 patients (44.0%) when at least 2 of 3 RT-PCR PSA assays per patient were positive. A much higher rate of RT-PCR PSA positivity was noted (77/116 patients, 66.3%) when any RT-PCR PSA positivity was considered. In 10 randomly selected cases the RT-PCR product was confirmed as PSA by deoxyribonucleic acid sequencing. Of 51 bone marrow RT-PCR positive cases 25 (49%) had organ confined disease and 26 (51%) had nonorgan confined disease. Similarly, bone marrow RT-PCR PSA was not associated with age, race, grade, pretreatment PSA or prostatic acid phosphatase value, clinical stage or margin status. However, the 2-year disease-free survival was 96.6% in RT-PCR negative patients versus 77.5% in RT-PCR positive patients (p = 0.054), and bone marrow RT-PCR PSA was an independent prognostic factor in multivariate analysis including PSA, Gleason grade and pathological stage. CONCLUSIONS: Bone marrow RT-PCR PSA positivity in this study did not predict pathological stage, grade or margin positivity as determined from whole mount prostate cancer specimens. Furthermore, no relationship with age, grade or serum markers and bone marrow RT-PCR PSA positivity was noted. However, bone marrow RT-PCR PSA was associated with early disease recurrence. Further studies and longer followup are warranted to define the metastatic potential of the PSA expressing cells in the bone marrow of prostate cancer patients.

Aged↗
PubMed1995

Surfactant protein gene expression in metastatic and micrometastatic pulmonary adenocarcinomas and other non-small cell lung carcinomas: detection by reverse transcriptase-polymerase chain reaction.

Reverse transcriptase (RT)-PCR with primers specific for surfactant protein A (SP-A), B (SP-B), C (SP-C), and D (SP-D) genes was applied to detect metastatic non-small cell lung carcinomas. Forty-one paratracheal and subcarinal lymph nodes obtained from 41 patients with non-small cell lung carcinomas, 11 lymph nodes from 11 patients with extrapulmonary adenocarcinomas, and eight control lymph nodes from patients without cancer were analyzed using RT-PCR. PCR products corresponding to SP-B gene products were found in all eight control lymph nodes, offering evidence of SP-B gene expression in cells of lymphatic tissue. SP-A, SP-C, and/or SP-D transcripts were detected in 11 (84.6%) of 13 lymph nodes with histologically identifiable metastases of pulmonary adenocarcinomas and in 10 (55.5%) of 18 lymph nodes that were tumor free on routine histological examination. These findings provide evidence of micrometastatic nodal involvement which remains undetectable by conventional light microscopy but can be evaluated by surfactant RT-PCR. Gene expression of SP-A and SP-C was restricted to metastatic pulmonary adenocarcinomas but SP-D gene activity has been also detected in two of four metastases of pulmonary large cell carcinomas, one adenosquamous carcinoma, and nine extrapulmonary adenocarcinomas as well.

Adenocarcinoma↗
PubMed2005

Determining suitable internal standards for mRNA quantification of increasing cancer progression in human breast cells by real-time reverse transcriptase polymerase chain reaction.

Real-time reverse transcriptase polymerase chain reaction is recognized as a highly sensitive and specific method for quantification of mRNA expression. SYBR green I dye simplifies the experimental design but introduces the need for specific controls to maintain high specificity. Due to this increased sensitivity, standards that may have been acceptable for normalization of less sensitive methods have been shown to vary considerably among cell lines, tissues, proliferative states, treatments, and developmental conditions and by degree of cancer progression. It has become evident that determination of suitable normalization standards is a requirement for the use of this method as it is applied toward any new experimental model. We have assessed the suitability of a number of commonly used standards for the normalization of mRNAs among a set of human breast cancer cell lines of increasing metastatic potential and have determined that 18S rRNA and beta-actin (ACTB) mRNA are both suitable for this purpose, with each having some limitations. 18S rRNA varies less among the cell lines but has a higher degree of random variability, while ACTB mRNA varies more among cell lines but has a lower degree of random variation.

Actins↗
PubMed1997

Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction.

A strategy for reverse transcriptase polymerase chain reaction (RT-PCR) using multiple primers was developed to detect and to differentiate the seven serotypes of foot-and-mouth disease virus (FMDV) simultaneously, quickly and accurately. The development of the test was carried out on virus isolates grown in tissue culture prior to cDNA synthesis and PCR using various sets of primers. Primers P33 and P32 were used for the consensus PCR to detect FMDV regardless of the serotype. Positive cDNA was assayed in two multi-primer PCR mixes containing type-specific primers capable of distinguishing between the seven serotypes. The serotype-specific primers were selected to correspond to regions of the genome coding for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus group. Multi-primer mix P33-P(A-C-O-ASIA 1) gave products of 732, 596, 402, 292 bp for the A,C,O and ASIA 1 serotypes, respectively, and no target products for South African Territories serotypes (SAT 1, SAT 2 and SAT 3). The multi-primer mix P33-P(A-C-O-ASIA 1) was also capable of detecting a mixture of two different serotypes. Multi-primer mix P1-P(SAT 1-3-2) gave products of 246, 201 and 75 bp for the SAT 1, SAT 3 and SAT 2 serotypes, respectively, and no specific products for serotypes A, C, O and ASIA 1. This is the first PCR assay to be described that differentiates between the SAT serotypes of FMDV. The method has been applied to 25 cell-culture-derived isolates of the SAT serotypes of FMDV and the results were totally compatible with the standard techniques for FMDV detection and serotyping.

Animals↗
PubMed1999

Improved methods using the reverse transcriptase polymerase chain reaction to detect tumour cells.

Reverse transcriptase polymerase chain reaction (RT-PCR) is increasingly used to detect small numbers of circulating tumour cells, though the clinical benefit remains controversial. The largest single contributing factor to the controversy of its value is the different approaches to sample processing. The aim of this study was to compare the sensitivity and reproducibility of RT-PCR for the detection of tumour cells after four commonly used different methods of sample processing. Using RT-PCR, one tumour cell spiked in 2 ml of whole blood was detected after analysis of separated mononuclear cell RNA, whole blood total or poly-A+ RNA. No false positives were identified with any method. However, the reproducibility of tumour cell detection was reduced after isolation of the mononuclear cell fraction. Only analysis of poly-A+ RNA had a sensitivity of 100% in all the cell spiking experiments. In patient blood samples, analysis of poly-A+ RNA increased the number of blood samples positive for tyrosine hydroxylase (TH) mRNA compared with those positive after analysis of total RNA. This may reflect high levels of cDNA reducing the efficiency of the PCR. Isolation of poly-A+ RNA increases the sensitivity and reproducibility of tumour cell detection in peripheral blood.

DNA↗
PubMed2001

Detection and clinical significance of occult tumour cells in colorectal cancer.

BACKGROUND: A variety of techniques have been employed for the detection of occult tumour cells in the blood, bone marrow and lymph nodes of patients with colorectal cancer. This review examines the methods used, results obtained and the clinical significance of studies in this field. METHODS: A Medline literature search was performed using the terms colorectal cancer, minimal residual disease, micrometastasis, polymerase chain reaction, reverse transcriptase polymerase chain reaction and immunocytochemistry; further references were obtained from key articles. RESULTS: Immunocytochemical examination of bone marrow is the benchmark for detecting clinically significant occult disease. Larger standardized studies are required to confirm the prognostic significance of molecular assays for the detection of tumour cells in blood and bone marrow. The prognostic significance of lymph node tumour cells detected by either immunohistochemical or molecular methods awaits further affirmation. CONCLUSION: Standardization of terminology and techniques used, combined with large prospective clinical studies, is required if detection of occult residual disease is to become a prognostic marker for recurrence in colorectal cancer.

Biomarkers, Tumor↗
PubMed1996

Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR).

The reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect contamination of Marek's disease (MD) vaccine with reticuloendotheliosis virus (REV). The env primers were used for the 1st RT-PCR to amplify the DNA fragments of REV-A and -T. The rel and env primers were used for nested-PCR to confirm the sites deleted from REV-T and REV-A. Specific amplification products were detected in the 1st RT-PCR with these primers. By nested PCR with the env and the rel primer pairs, the products originating from REV-A and -T were identified. This system, using the env primer pairs, showed a specific amplification with several REV strains (REV-T, DE, CE, KI and 0202), but no amplified product was detected with MDV, NDV, IBV or ILTV. The 1st RT-PCR detected the virus in a concentration of 10(3) in 50% fluorescent antibody infectious dose per ml (FAID50/ml) and the nested PCR detected 10(1) FAID50/ml virus. The sensitivity of the RT-PCR system was found to be higher than that of the FA assay. This system provides a rapid, sensitive and specific method for detection of contamination of MD vaccines with REV-RNA, and it may be applied for quality control of live vaccines.

Animals↗
PubMed1996

Identification of carcinoembryonic antigen-producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction.

BACKGROUND: Application of the reverse transcriptase polymerase chain reaction (RT-PCR) to identification of circulating tumour cells in colorectal cancer. AIMS: To assess whether circulating malignant cells in patients with colorectal liver metastasis could be identified by RT-PCR recognition of mRNA coding for the tumour marker carcinoembryonic antigen (CEA). PATIENTS: A total of 31 with colorectal liver metastases and 22 no-cancer controls. METHODS: Specific cDNA primers for CEA transcripts were used to apply RT-PCR to tissue biopsy specimens, colon carcinoma cell lines, and peripheral blood samples from patients with colorectal liver metastases. A strongly CEA-expressive HT115 colorectal carcinoma cell line was used to spike blood samples from no-cancer control subjects. RESULTS: The limit for detection of CEA cDNA by Southern blotting using HT115 cells was 50 cells per 14 ml of spiked blood. There was a significant difference (p = 0.007) in RT-PCR positive expression between patients with liver metastasis (26/31) compared with controls (5/22). There was no significant relation between the prevalence of CEA cDNA amplification and serum CEA level or metastasis volume in patients with liver metastasis. CONCLUSIONS: This is the first study to suggest that identification of circulating colorectal cancer cells using RT-PCR for detection of CEA cDNA is feasible.

Biomarkers, Tumor↗
PubMed1996

[Detection of breast cancer micrometastases in lymph nodes by amplification of keratin 19 mRNA with reverse-transcriptase polymerase chain reaction].

Keratin 19 mRNA reverse-transcriptase polymerase chain reaction (K 19 RT-PCR) was compared with histological examination in the detection of breast cancer micrometastases in axillary lymph nodes. Sixty-three axillary lymph nodes, which were obtained from 23 breast cancer patients, were bisected. One half was studied by conventional histological analysis of hematoxylin and eosin sections. Total RNA was extracted from the other half and subjected to K 19 RT-PCR. In all the ten lymph nodes, which were histologically metastasis-positive, K 19 mRNA was detected by RT-PCR. Of the 53 histologically negative lymph nodes, five (9%) lymph nodes were found to express K 19 mRNA, indicating the presence of micrometastases which could be detected by RT-PCR but not by a histological examination. These results demonstrate the usefulness of K 19 RT-PCR in the detection of breast cancer micrometastases in lymph nodes.

Axilla↗
PubMed1997

Quantitative analysis of human multidrug resistance 1 (MDR1) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay.

We have established competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay for the quantification of MDR1 mRNA encoding P-glycoprotein (P-gp) by analyzing leukemia sublines of MOLT-3 with various expression of MDR1. The expression was quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shares the MDR1 primer sequences with the cellular MDR1 mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments. The amounts of MDR1 mRNA measured by the assay were accurate and reproducible over wide range, and were determined as 31.6, 100, and 316 amol/microgram total RNA in MOLT-3/TMQ70, MOLT-3/ TMQ800, and MOLT-3/VCR1,000, respectively. The relative ratio of MDR1 mRNA measured by the competitive RT-PCR among three sublines was similar to that of MDR1 transcript determined by Northern analysis (1:4:12) and to that of P-gp measured by flow cytometry (FCM) analysis. In mononuclear cells from patients with leukemia, MDR1 mRNA could be sufficiently quantified by the competitive RT-PCR established, while FCM assay could scarcely detet P-gp. This study demonstrated that the competitive RT-PCR assay using heterologous competitor RNA is a rapid, reliable, and non-radioactive procedure and is acceptable for the evaluation of MDR1 expression in clinical samples.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Prolonged measles virus shedding in human immunodeficiency virus-infected children, detected by reverse transcriptase-polymerase chain reaction.

A reverse transcriptase-polymerase chain reaction assay was used to detect measles virus RNA in peripheral blood mononuclear cells, urine, and nasopharyngeal specimens from Zambian children during hospitalization and approximately 1-2 months after discharge. Of 47 children, 29 (61.7%) had prolonged measles virus shedding, as defined by detection of measles virus RNA in > or =1 specimen obtained 30-61 days after rash onset. Ten (90.9%) of 11 human immunodeficiency virus (HIV)-infected children had prolonged measles virus shedding, compared with 19 (52.8%) of 36 HIV-uninfected children (P=.02). Prolonged measles virus shedding did not correlate with levels of measles virus-specific antibody. HIV-infected children with measles may have a prolonged infectious period that potentially enhances measles virus transmission and hinders measles control.

Adolescent↗
PubMedDate not supplied

Detection of occult tumor cells in peripheral blood from patients with small cell lung cancer by reverse transcriptase-polymerase chain reaction.

The reverse transcriptase-polymerase chain reaction (RT-PCR) of tumor-specific or -associated genes is a sensitive assay for detecting a minimal number of tumor cells in peripheral blood (PB) or bone marrow (BM). In this study, we determined whether mRNA of bombesin receptors is detectable in PB or peripheral blood progenitor cell (PBPC) samples from patients with small cell lung cancer. Among three bombesin-like peptide receptors, we used the neuromedin B receptor (NMB-R) gene as a target, because of the most frequent expression on SCLC cell lines. The lower limit of detection was one tumor cell in one million normal PB cells and there was no detection in normal PB or BM cells unlike a cytokeratin 19 gene. The NMB-R gene was detected in 14 (31.8%) of 44 PB samples from patients with SCLC at diagnosis and 2 (15.4%) of 13 samples of PBPC collected during a recovery phase after chemotherapy followed by administration of G-CSF (filgrastim). At diagnosis, patients whose PB was positive for the NMB-R gene had a significantly shorter survival than those who were negative. Our observation suggests that this assay may be useful in diagnosing metastatic disease and monitoring minimal residual disease in patients with SCLC.

Aged↗
PubMed2002

Detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis.

Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proved useful for the detection of mouse hepatitis virus (MHV) and rat coronavirus (RCV) in acutely infected animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. Consequently, a fluorogenic nuclease RT-PCR assay specific for rodent coronaviruses was developed. Primer and probe sequences were selected from the viral genome segment that encodes the membrane (M) protein that is highly conserved among rodent coronaviruses. Use of the fluorogenic nuclease RT-PCR detected all strains of MHV and RCV that were evaluated, but did not detect other RNA viruses that naturally infect rodents. Use of the assay detected as little as two femtograms of in vitro transcribed RNA generated from cloned amplicon, and when compared directly with mouse antibody production tests, had similar sensitivity at detecting MHV-A59 in infected cell culture lysates. Finally, use of the assay detected coronavirus RNA in tissues, cage swipes, and feces obtained from mice experimentally infected with MHV, and in tissues and cage swipes obtained from rats naturally infected with RCV. These results indicate that the fluorogenic nuclease RT-PCR assay should provide a potentially high-throughput, PCR-based method to detect rodent coronaviruses in infected rodents and contaminated biological materials.

Animals↗
PubMedDate not supplied

Amplification of avian reovirus RNA using the reverse transcriptase-polymerase chain reaction.

A reverse transcriptase-polymerase chain reaction method was developed for the detection of avian reovirus. The origin of primers was from the S1 gene of the avian reovirus genome. A reovirus-specific 532-base pair cDNA product was amplified by these primers from six reference strains and 23 field isolates of avian reoviruses, but not from seven different avian pathogenic viruses and bacteria. As little as 1 pg of avian reovirus RNA was detected using gel electrophoresis and Southern blot hybridization.

Animals↗