scholarly journals Monitoring Shedding of Five Genotypes of RotaTeq Vaccine Viruses by Genotype-Specific Real-Time Reverse Transcription-PCR Assays

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Yuki Higashimoto ◽  
Masaru Ihira ◽  
Yu Miyazaki ◽  
Ayumi Kuboshiki ◽  
Sayaka Yoshinaga ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Cross Reactivity ◽  
Rt Pcr ◽  
Fecal Shedding ◽  
Reverse Transcription Pcr ◽  
Coefficients Of Variation ◽  
Long Time ◽  
Stool Samples ◽  
Pcr Assays

ABSTRACTRotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

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 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 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 Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

Detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in Henan Province, China, using real-time reverse transcription PCR

2013 ◽  
Vol 62 (7) ◽  
pp. 1060-1064 ◽  
Author(s):  
Xueyong Huang ◽  
Licheng Liu ◽  
Yanhua Du ◽  
Hongxia Ma ◽  
Yujiao Mu ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Henan Province ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Reverse Transcription Pcr

A novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome (FTLS) was discovered in Henan Province, China. Here, we report the development of an assay for this novel bunyavirus based on real-time reverse transcription PCR (RT-PCR). The assay exhibited high sensitivity and specificity without cross-reactivity towards 13 other viruses that cause similar symptoms. To evaluate the performance of this assay in detecting clinical samples, we analysed 261 serum samples from patients in Henan Province between 2007 and 2010. Of these samples, 91.95 % were bunyavirus positive. Compared with serological assays, the real-time PCR assay was much more sensitive in identifying infected patients 1 to 7 days after the onset of symptoms.

scholarly journals Genogroup I and II Noroviruses Detected in Stool Samples by Real-Time Reverse Transcription-PCR Using Highly Degenerate Universal Primers

2004 ◽  
Vol 70 (12) ◽  
pp. 7179-7184 ◽  
Author(s):  
Gary P. Richards ◽  
Michael A. Watson ◽  
Rebecca L. Fankhauser ◽  
Stephan S. Monroe
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Universal Primers ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Reverse Transcription Pcr ◽  
Wide Range ◽  
Genetic Clusters ◽  
Stool Samples

ABSTRACT Genogroup I noroviruses from five genetic clusters and genogroup II noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse transcription-PCR (RT-PCR) with SYBR Green detection. Two degenerate primer sets, designated MON 431-433 and MON 432-434, were designed from consensus sequences from the major clusters of norovirus based on the RNA-dependent RNA polymerase region of the norovirus genome. Viruses were extracted from stool samples within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semiquantitative results by means of the cycle threshold data and dilution endpoint standard curves. Presumptive product verification was achieved by evaluation of first-derivative melt graphs. Multiple clusters of noroviruses were identified simultaneously in a multiplex fashion by virtue of slight differences in melting temperature. The detection of 13 different genetic clusters suggests that the MON primers may serve as universal primers for most, if not all, of the noroviruses in a multiplex assay. Our technique provides a framework for broad application of real-time RT-PCR in clinical, environmental, and food testing laboratories for a wide range of noroviruses.

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