scholarly journals Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

2005 ◽  
Vol 71 (4) ◽  
pp. 1870-1875 ◽  
Author(s):  
Narayanan Jothikumar ◽  
James A. Lowther ◽  
Kathleen Henshilwood ◽  
David N. Lees ◽  
Vincent R. Hill ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Nested Pcr ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Specificity And Sensitivity ◽  
Routine Monitoring ◽  
The Family ◽  
One Step ◽  
Stool Specimens ◽  
Pcr Assays

ABSTRACT Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

scholarly journals Circulating Prostate Tumor Cells Detected by Reverse Transcription-PCR in Men with Localized or Castration-Refractory Prostate Cancer: Concordance with CellSearch Assay and Association with Bone Metastases and with Survival

2009 ◽  
Vol 55 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Pauliina Helo ◽  
Angel M Cronin ◽  
Daniel C Danila ◽  
Sven Wenske ◽  
Rita Gonzalez-Espinoza ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Metastases ◽  
Healthy Volunteers ◽  
Real Time ◽  
Tumor Cells ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Reverse transcription-PCR (RT-PCR) assays have been used for analysis of circulating tumor cells (CTCs), but their clinical value has yet to be established. We assessed men with localized prostate cancer or castration-refractory prostate cancer (CRPC) for CTCs via real-time RT-PCR assays for KLK3 [kallikrein-related peptidase 3; i.e., prostate-specific antigen (PSA)] and KLK2 mRNAs. We also assessed the association of CTCs with disease characteristics and survival. Methods: KLK3, KLK2, and PSCA (prostate stem cell antigen) mRNAs were measured by standardized, quantitative real-time RT-PCR assays in blood samples from 180 localized-disease patients, 76 metastatic CRPC patients, and 19 healthy volunteers. CRPC samples were also tested for CTCs by an immunomagnetic separation system (CellSearch™; Veridex) approved for clinical use. Results: All healthy volunteers were negative for KLK mRNAs. Results of tests for KLK3 or KLK2 mRNAs were positive (≥80 mRNAs/mL blood) in 37 patients (49%) with CRPC but in only 15 patients (8%) with localized cancer. RT-PCR and CellSearch CTC results were strongly concordant (80%–85%) and correlated (Kendall τ, 0.60–0.68). Among CRPC patients, KLK mRNAs and CellSearch CTCs were closely associated with clinical evidence of bone metastases and with survival but were only modestly correlated with serum PSA concentrations. PSCA mRNA was detected in only 7 CRPC patients (10%) and was associated with a positive KLK mRNA status. Conclusions: Real-time RT-PCR assays of KLK mRNAs are highly concordant with CellSearch CTC results in patients with CRPC. KLK2/3-expressing CTCs are common in men with CRPC and bone metastases but are rare in patients with metastases diagnosed only in soft tissues and patients with localized cancer.

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals An Integrated Diagnostic Approach for Citrus Exocortis Viroid

2021 ◽  
Author(s):  
Chih-Hsu Lin ◽  
Ting-Hsuan Hung ◽  
I Hu ◽  
Ta-Hsin Ku ◽  
Chun-Yi Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Severe Disease ◽  
Blot Hybridization ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Cultivar ◽  
Dot Blot Hybridization ◽  
Reverse Transcription Pcr ◽  
One Step

Abstract BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

scholarly journals Comparison of Four Serological Methods and Two Reverse Transcription-PCR Assays for Diagnosis and Surveillance of Zika Virus Infection

2018 ◽  
Vol 56 (3) ◽  
Author(s):  
Angel Balmaseda ◽  
José Victor Zambrana ◽  
Damaris Collado ◽  
Nadezna García ◽  
Saira Saborío ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Zika Virus ◽  
Clinical Manifestations ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Emerging Infections ◽  
Rt Pcr ◽  
Convalescent Phase ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

ABSTRACTZika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barré syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.

scholarly journals One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus

2019 ◽  
Vol 31 (6) ◽  
pp. 909-912 ◽  
Author(s):  
Chunyan Jiang ◽  
Haijian He ◽  
Chaoying Zhang ◽  
Xiaoju Zhang ◽  
Jianfeng Han ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Porcine Epidemic Diarrhea Virus ◽  
Detection Method ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Reverse Transcription Pcr ◽  
Porcine Sapovirus ◽  
One Step ◽  
Porcine Epidemic Diarrhea ◽  
Porcine Sapelovirus

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 μM for PEDV, 0.15 μM for PSV, and 0.2 μM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR–based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

scholarly journals Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

2007 ◽  
Vol 53 (11) ◽  
pp. 1899-1905 ◽  
Author(s):  
Marit Kramski ◽  
Helga Meisel ◽  
Boris Klempa ◽  
Detlev H Krüger ◽  
Georg Pauli ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hantaan Virus ◽  
Hantavirus Infection ◽  
Sequence Information ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

scholarly journals Duplex Reverse Transcription-PCR Followed by Nested PCR Assays for Detection and Identification of Brazilian Alphaviruses and Flaviviruses

2005 ◽  
Vol 43 (2) ◽  
pp. 696-702 ◽  
Author(s):  
R. V. d. M. Bronzoni ◽  
F. G. Baleotti ◽  
R. M. Ribeiro Nogueira ◽  
M. Nunes ◽  
L. T. Moraes Figueiredo
Keyword(s):  
Reverse Transcription ◽  
Nested Pcr ◽  
Reverse Transcription Pcr ◽  
Detection And Identification ◽  
Pcr Assays ◽  
Nested Pcr Assays
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