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Results for “Reverse Transcriptase Polymerase Chain Reaction”

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PubMed2005

Rapid detection of chikungunya virus in laboratory infected Aedes aegypti by Reverse-Transcriptase- Polymerase Chain Reaction (RT-PCR).

A study of chikungunya virus was carried out to establish Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) as a rapid detection technique of the virus. The susceptibility of lab-colonized Aedes aegypti to chikungunya virus was also determined. Artificial membrane feeding technique was used to orally feed the mosquitoes with a human isolate of chikungunya virus. A total of 100 fully engorged female Ae. aegypti were obtained and maintained for 7 days. Seventy of them survived and then pooled at 10 individuals per pool. Total RNA was extracted from the samples and RT-PCR amplifications were carried out. Five out of 7 pools showed positive PCR band at 350-bp, indicating Ae. aegypti is a potential vector of chikungunya virus. The minimum infection rate (MIR) was 71% within these laboratory colonies. RT-PCR is a sensitive technique that is useful in detecting infected mosquitoes in epidemic areas. This technique can de used as a rapid detection method and provide an early virologic surveillance systems of chikungunya virus infected mosquitoes.

Aedes↗
PubMed1999

Demonstration of viable Chlamydia pneumoniae in atherosclerotic plaques of carotid arteries by reverse transcriptase polymerase chain reaction.

The presence of Chlamydia pneumoniae in atheromas has been demonstrated in several studies. Culture of the organism from arterial tissue has been difficult. We report the use of a reverse transcriptase polymerase chain reaction to detect viable Chlamydia pneumoniae in carotid atheromas. We analyzed 30 patients (14 females, mean age 69.6 +/- 8.8 years) who underwent surgery for the removal of atherosclerotic plaques from carotid arteries. During surgery, samples of lingual vein and superior thyroideal artery were also taken. We applied two molecular biology techniques to the carotid plaques on lingual vein or thyroideal artery samples: 1) polymerase chain reaction (PCR) and 2) reverse transcriptase-PCR (RT-PCR) for the detection of bacterial mRNA, employing PCR primers designed to detect a fragment of the 16S rRNA gene. Blood samples were obtained from the patients for determination of Chlamydia pneumoniae IgG, IgA, and IgM antibody titers by a microimmunofluorescence technique. The results of the present study confirmed the presence of viable Chlamydia pneumoniae in atheromas and support the hypothesis that the organism may be an active factor in the pathogenesis of atherosclerosis.

Aged↗
PubMed2003

Five years' experience of reverse-transcriptase polymerase chain reaction in daily diagnosis of enterovirus and rhinovirus infections.

To determine the efficiency of reverse-transcriptase polymerase chain reaction (RT-PCR) assays currently used in diagnosing enterovirus and rhinovirus infections, we compared results obtained with RT-PCR methods, which detect both enteroviruses and rhinoviruses simultaneously, with results obtained by conventional virus isolation. Both tests were performed on 591 specimens: 38 samples (6%) had positive results by both RT-PCR and isolation, 90 samples (15%) had positive results by RT-PCR only, and 7 samples (1%) had positive results only by virus isolation. In conclusion, RT-PCR was superior in rapidity and sensitivity to virus isolation and is recommended as the primary diagnostic tool for enterovirus and rhinovirus infections.

Enterovirus↗
PubMed1997

The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19.

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19. RT-PCR was done by two 40-cycle rounds using nested primers. In initial experiments, RT-PCR was shown to be capable of detecting one tumour cell in one million normal bone marrow cells, which was at least 10 times more sensitive than immunohistochemistry, while retaining specificity. Five per cent of the peripheral blood and 22% of the bone marrow samples contained K19 positive cells by immunohistochemistry staining. Using RT-PCR, these proportions increased to 25% and 35%, respectively. This represents a significantly greater detection frequency (P < 0.001 and P = 0.03, respectively). RT-PCR for K19 is a more sensitive method for detecting micrometastases in patients with primary breast cancer when compared with immunohistochemistry.

Adult↗
PubMed1996

Evaluation of reverse transcriptase polymerase chain reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance.

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was evaluated for the detection of eastern equine encephalomyelitis virus (EEEV). EEEV was detected by amplification of a 416-bp PCR product from within the E2 gene. Internal restriction endonuclease digestion and hybridizations to EEEV RNA demonstrated that the PCR product was amplified from EEEV. PCR amplifications from serial dilutions of an EEEV isolate identified by a neutralization test and titered by an infectious assay in cell culture indicated that this RT-PCR assay detected viral RNA at concentrations below 1 plaque forming unit(PFU) per reaction. The performance of the PCR assay in detection of EEEV was compared with an infectious assay detection procedure (IA/IFA) as part of the New Jersey 1993 vector surveillance program. During 1993, 7,007 field-collected Culiseta melanura (Coquillett) were assayed in 522 pools by both RT-PCR and IA/IFA. EEEV was detected in 95 pools by RT-PCR and 17 pools by IA/IFA; all IA/IFA positive pools were also positive by RT-PCR. During the 1993 field season, RT-PCR consistently detected virus at enzootic foci earlier that IA/IFA and in greater numbers of mosquito pools. The data indicated that viral RNA may be present earlier and in more mosquitoes than indicated by IA/IFA.

Aedes↗
PubMed1990

Use of reverse transcriptase polymerase chain reaction to monitor expression of intronless genes.

Our data demonstrate the use of the reverse transcriptase polymerase chain reaction (RT-PCR) technique to study mRNA expression of genes that are devoid of introns. We have developed conditions that eliminate the false positives that can result from any preexisting DNA and that could confuse the interpretation of results. This modification (DNase pretreatment under specified conditions) ensures that the product resulting from RT-PCR is due to amplification of cDNA that has been synthesized during the reverse transcriptase reaction. Our results illustrate and emphasize the importance of including both a DNase pretreatment and a minus RT control. Using this modified procedure, our data illustrate clearly the ability of this protocol to demonstrate the presence of very low levels of olfactory marker protein (OMP) mRNA in three non-olfactory rat brain regions (cerebellum, thalamus/hypothalamus and cerebral hemispheres) where OMP mRNA was previously unknown. These data confirm a prior report of the ectopic expression of OMP immunoreactivity in these locations and indicate for the first time the "illegitimate" expression of extremely low levels of OMP mRNA in a non-neural tissue. Finally, this modification of the RT-PCR procedure will now permit the study of expression of specific, rare, mRNA molecules in the absence of any prior knowledge of the structure of their genes of origin.

Animals↗
PubMed2006

Coxsackievirus B3 in human milk.

Coxsackievirus B3 can cause severe neonatal disease with high mortality. We present the first report of detection of coxsackievirus B3 in the mothers' milk of 2 severely infected neonates by culture and reverse transcriptase-polymerase chain reaction. Reverse transcriptase-polymerase chain reaction is a rapid and sensitive tool to detect coxsackievirus B3 in breast milk within few hours. By 12 to 14 days after onset of symptoms, coxsackievirus B3 could not be detected in the breast milk of the symptomatic mothers.

Adult↗
PubMed2006

Prospective multi-institutional study of reverse transcriptase polymerase chain reaction for molecular staging of melanoma.

PURPOSE: To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream. PATIENTS AND METHODS: In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma > or = 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant-DFS (DDFS), and overall survival (OS) were analyzed. RESULTS: A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR-positive (n = 620) and RT-PCR-negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood. CONCLUSION: In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

Adult↗
PubMed1995

Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis.

A limitation to date of reverse transcriptase polymerase chain reactions (RT-PCRs) for the detection of small, round structured viruses (SRSVs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (N1/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)-labelled oligonucleotides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from approximately 5% of amplified strains did not hybridise. The N1/E3 primer pair were shown to be SRSV-specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology. Hybridisation of PCR products with the individual oligonucleotides relating to SRSV SPIEM types UK 1-4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT-PCR and offers the potential for direct detection in food and environmental samples.

Animals↗
PubMed1998

The value of a reverse transcriptase polymerase chain reaction assay in preoperative staging and followup of patients with prostate cancer.

PURPOSE: The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an extremely sensitive technique of detecting cells expressing prostate specific antigen (PSA). Controversy exists regarding the ability of peripheral blood PSA RT-PCR testing to reflect pathological staging or treatment outcome. We examine the phenomenology of RT-PCR results in patients with prostate cancer, with particular emphasis on the RT-PCR test before and after radical prostatectomy, and correlations with pathological staging and treatment outcome. MATERIALS AND METHODS: Peripheral blood was obtained from a wide variety of patients with and without prostate cancer, including before and after radical prostatectomy. After ribonucleic acid isolation, complementary deoxyribonucleic acid was generated and amplified with a hot-start technique. RT-PCR results were compared with pathological stage, Gleason score, tumor volume and disease-free status. Correlations between preoperative and postoperative RT-PCR tests were also made. RESULTS: The RT-PCR test was positive in 1 of 56 controls (1.8%) without suspicion of prostate cancer. A positive test was obtained in 12 of 65 men (18.5%) with a suspicion of prostate cancer but a negative biopsy. Before radical prostatectomy a positive test was obtained in 13 of 75 men (17.3%) with pT2 disease versus 10 of 46 (21.7%) with pT3 disease. There was no significant difference in serum PSA, Gleason score or tumor volume between the men with positive or negative results. With repetitive testing an increasing percentage of men had at least 1 positive test preoperatively. With a median followup of 8 months 6 of the 7 patients in whom radical prostatectomy failed had had negative RT-PCR before treatment. Of patients with known metastatic disease or failed primary treatment a positive test was obtained in 32 to 75%. Radical prostatectomy and prostate needle biopsy appeared to have a negligible effect on RT-PCR tests immediately following these procedures. Following radical prostatectomy results were variable but many men who are RT-PCR positive preoperatively become RT-PCR negative postoperatively. CONCLUSIONS: The PSA RT-PCR test in our laboratory cannot be used preoperatively to predict pathological stage of prostate cancer or treatment failure. Most cases that are positive preoperatively become negative postoperatively. While increasing tumor burden increases the likelihood of positive tests, there appears to be significant sampling error associated with the use of this test in the peripheral blood.

Adult↗
PubMed1999

Sensitive detection of rare Ewing's sarcoma cells in peripheral blood by reverse transcriptase polymerase chain reaction.

Disseminated disease is very important in the clinical assessment of pediatric sarcomas. Several reports suggest that reverse transcriptase polymerase chain reaction (RT-PCR) holds great promise in the early staging of cancer patients in general. However, the complexities of these protocols hamper adequate standardization, and their application as routine diagnostic tools has been difficult. The aim of this study is to assess the actual minimal number of tumor cells that may be detected by RT-PCR in a blood sample. Specific tumor cell dilutions from a Ewing's sarcoma cell line reconstituted in peripheral blood from healthy individuals were "ficolled" and submitted to RNA extraction for cDNA preparation and PCR amplification of the t(11-22) (q24;q12) fusion transcript. After PCR amplification, we were able to detect the EWS/FI-1 chimeric gene product at a dilution of 10 tumor cells per 1 or 2 mL of blood. Our simple method supports a role for routine clinical use of RT-PCR in the detection of circulating Ewing's sarcoma cells.

Artificial Gene Fusion↗
PubMed2002

Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reverse-transcriptase polymerase chain reaction analysis.

Somatostatin receptors (SSTRs) have been extensively mapped in human tumors by means of autoradiography, reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). We analyzed the SSTR type 1-5 expression by means of RT-PCR and/or IHC in a series of 81 functioning and non-functioning gastroenteropancreatic (GEP) endocrine tumors and related normal tissues. Moreover, we compared the results with clinical, pathological and hormonal features. Forty-six cases (13 intestinal and 33 pancreatic) were studied for SSTR 1-5 expression using RT-PCR, IHC with antibodies to SSTR types 2, 3, 5 and ISH for SSTR2 mRNA. The vast majority of tumors expressed SSTR types 1, 2, 3 and 5, while SSTR4 was detected in a small minority. Due to the good correlation between RT-PCR and IHC data on SSTR types 2, 3, and 5, thirty-five additional GEP endocrine tumors were studied with IHC alone. Pancreatic insulinomas had an heterogeneous SSTR expression, while 100% of somatostatinomas expressed SSTR5 and 100% gastrinomas and glucagonomas expressed SSTR2. Pre-operative biopsy material showed an overlapping immunoreactivity with that of surgical specimens, suggesting that the SSTR status can be detected in the diagnostic work-up. It is concluded that SSTRs 1-5 are heterogeneously expressed in GEP endocrine tumors and that IHC is a reliable tool to detect SSTR types 2, 3 and 5 in surgical and biopsy specimens.

Adult↗
PubMed1994

Arginine vasotocin gene expression in neuroendocrine, reproductive and gastrointestinal tissues of the domestic fowl: detection by reverse transcriptase polymerase chain reaction.

Arginine vasotocin gene transcripts in various tissues of the domestic fowl were detected by reverse transcriptase polymerase chain reaction followed by Southern blot analysis using a 209 bp fragment from the 3'-region of a cDNA encoding chicken arginine vasotocin as the probe. Relatively strong signals were observed with hypothalamic, adenohypophysial and proventricular RNA as the starting material. Lesser signals were obtained from RNA isolated from shell gland, adrenal gland, post-ovulatory follicles and ovarian thecal cells. Arginine vasotocin gene transcripts were undetectable in the posterior pituitary gland, small intestine and large intestine. These results suggest that in addition to its well-known antidiuretic and oxytocic actions, arginine vasotocin may act as a local neuromodulator or mediator and have other important autocrine or paracrine actions in non-hypothalamic tissues.

Animals↗
PubMed1999

Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.

The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the sequence of reference strain Bucyrus, the analysed samples were 78-100% identical and showed a 84-97% nucleotide identity with Bucyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extracted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that this could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be sensitive, specific and above all directly applicable to semen. Additionally, the short time needed for the overall procedure makes this method suitable for diagnostic purposes.

Animals↗
PubMed2000

Detection of circulating breast cancer cells by reverse transcriptase polymerase chain reaction (RT-PCR).

The confounding problem in treatment of breast cancer is the metastasis of breast tumour. Reverse transcriptase polymerase chain reaction (RT-PCR) has been recently used in the detection of circulating breast cancer cells. This review reports on the development of this assay as well as its advantages and disadvantages. We feel that cytokeratin 20 and beta -human chorionic gonadotropin (hCG) mRNA are the best markers for the detection of circulating breast cancer cells. We suggest that the multiple RNA marker RT-PCR assay can help to increase both sensitivity and specificity of detection, and that quantitative RT-PCR assay is more effective than the qualitative assay in the detection of circulating breast cancer cells.

Biomarkers, Tumor↗
PubMed2002

Detection of disseminated cancer cells in bone marrow of gastric cancer using real time quantitative reverse transcriptase polymerase chain reaction.

Bone marrow is a prognostically relevant indicator organ for micrometastasis. In the present study, real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect disseminated gastric cancer cells in bone marrow. We compared CEA, CK18 and CK20 expression using four gastric cancer cell lines and three normal tissue cell lines in order to select the most appropriate marker for detection of disseminated gastric cancer cell in bone marrow. CK20 proved to be the most promising marker since the expression level of normal cell lines was extremely low and about 50--100-fold differences were found between gastric carcinoma cell lines and normal tissue cell lines. We also screened bone-marrow RNA of 47 patients with gastric cancers, using this system. Among the three markers we tested, with only about CK20 could we find that 27 of 47 patients were positive. Though long-term clinical follow up studies are needed to evaluate the clinical significance of this method, real time quantitative RT-PCR is sensitive and quantitative for detection of micrometastasis in bone marrow.

Adenocarcinoma↗
PubMed2003

Detection of Reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction.

Reovirus type 3 (Reo-3) can infect numerous rodent species and induces the clinical syndrome 'oily skin disease' in neonatal mice, and is a common contaminant of biological materials. The reverse transcriptase polymerase chain reaction (RT-PCR) assay has proven useful for the detection of Reo-3 in rodents and contaminated biological materials. Fluorogenic nuclease reverse transcriptase polymerase chain reaction assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, an fnRT-PCR assay specific for Reo-3 was developed by targeting primer and probe sequences to a unique region of the Reo-3 M3 gene. The fnRT-PCR detected both strains of Reo-3 (Dearing and Abney), but did not detect Reovirus types 1 or 2, other viruses in the family Reoviridae, or other RNA viruses that naturally infect rodents. The fnRT-PCR detected less than 1 fg of target template and detected viral RNA in tissues obtained from mice experimentally infected with Reo-3. The assay also displayed comparable sensitivity when compared to the mouse antibody production test commonly used to detect viral contamination of biological materials. In conclusion, this fnRT-PCR assay offers a potentially high-throughput diagnostic assay for detecting Reo-3 RNA in infected mice and contaminated biological materials.

Animals↗