scholarly journals Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

2016 ◽  
Vol 54 (7) ◽  
pp. 1766-1773 ◽  
Author(s):  
Monika Simmons ◽  
Todd Myers ◽  
Carolina Guevara ◽  
Donald Jungkind ◽  
Maya Williams ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Sensitivity And Specificity ◽  
Internal Control ◽  
Chikungunya Virus ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Denv Serotypes ◽  
Reverse Transcriptase Pcr ◽  
One Step

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), andin vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

scholarly journals Development of a Sensitive Real-Time Reverse Transcriptase PCR Assay with an Internal Control to Detect and Quantify Chikungunya Virus

2007 ◽  
Vol 53 (8) ◽  
pp. 1408-1414 ◽  
Author(s):  
Philippe Laurent ◽  
Karin Le Roux ◽  
Philippe Grivard ◽  
Gérard Bertil ◽  
Florence Naze ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Internal Control ◽  
Chikungunya Virus ◽  
Indian Subcontinent ◽  
False Negative ◽  
Western Indian Ocean ◽  
Absolute Quantification ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr

Abstract Background: The chikungunya virus (CHIKV; Alphavirus, Togaviridae) has emerged in the south Western Indian Ocean since early 2005. A major outbreak of CHIKV infection occurred in Réunion Island, where the virus is transmitted by Aedes albopictus mosquitoes. Facing an outbreak of unprecedented magnitude, we developed a rapid, sensitive, and reliable assay for the detection and quantification of CHIKV in plasma samples. Methods: A dual-color TaqMan 1-step reverse transcriptase PCR assay was developed in a LightCycler 2.0 system. A coextracted and coamplified chimerical RNA sequence was used as an internal control (IC) to eliminate false-negative results. The CHIKV-specific and IC probes were labeled with 6-carboxyfluorescein (530 nm) and the wide span dye DYXL (705 nm), respectively, eliminating the need for color compensation. A synthetic RNA was used as an external calibrator for CHIKV absolute quantification. Results: The detection limit was 350 copies/mL (3 copies/capillary). A further improvement to ∼40 copies/mL was obtained by use of a larger volume of plasma. The assay specificity was confirmed in vitro and in silico. CHIKV in 343 patients was present at viral loads >108 copies/mL, mainly in newborns and seniors >60 years old. Long viremic phases of up to 12 days were seen in 6 patients. Conclusions: The assay is rapid, CHIKV-specific, and highly sensitive, and it includes an IC. It proved useful to detect and quantify CHIKV during the Réunion Island epidemic. The assay might be applicable to other CHIKV epidemics, especially in the Indian subcontinent, where an extensive outbreak is ongoing.

scholarly journals Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR

1998 ◽  
Vol 36 (9) ◽  
pp. 2634-2639 ◽  
Author(s):  
Eva Harris ◽  
T. Guy Roberts ◽  
Leila Smith ◽  
John Selle ◽  
Laura D. Kramer ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dengue Fever ◽  
Internal Control ◽  
Extraction Procedure ◽  
Rna Degradation ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Single Tube ◽  
Dengue Viruses

In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.

scholarly journals Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of Zika virus and Chikungunya virus

2017 ◽  
Vol 90 (3) ◽  
pp. 389-396 ◽  
Author(s):  
Si-Qing Liu ◽  
Xiao Li ◽  
Cheng-Lin Deng ◽  
Zhi-Ming Yuan ◽  
Bo Zhang
Keyword(s):  
Real Time ◽  
Zika Virus ◽  
Chikungunya Virus ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

scholarly journals Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252789
Author(s):  
Yukiko Nakura ◽  
Heng Ning Wu ◽  
Yuya Okamoto ◽  
Muneyuki Takeuchi ◽  
Koichiro Suzuki ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Dna Polymerase ◽  
Internal Control ◽  
Leukemia Virus ◽  
Rt Pcr ◽  
Thermotoga Petrophila ◽  
One Step ◽  
Thermostable Dna Polymerase

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

One-step Multiplex RT-PCR Assay for the Detection of Peste des petits ruminants Virus in Clinical Samples

2006 ◽  
Vol 30 (6) ◽  
pp. 655-666 ◽  
Author(s):  
V. Balamurugan ◽  
A. Sen ◽  
P. Saravanan ◽  
R. P. Singh ◽  
R. K. Singh ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Peste Des Petits Ruminants ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

scholarly journals Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

2015 ◽  
Vol 53 (8) ◽  
pp. 2641-2647 ◽  
Author(s):  
Todd N. Wylie ◽  
Kristine M. Wylie ◽  
Richard S. Buller ◽  
Maria Cannella ◽  
Gregory A. Storch
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Limit Of Detection ◽  
Respiratory Illness ◽  
The United States ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr ◽  
Enterovirus D68

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

scholarly journals Potential Point-of-Care Testing for Dengue Virus in the Field

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Wei-Kung Wang ◽  
Duane J. Gubler
Keyword(s):  
Dengue Virus ◽  
Diagnostic Test ◽  
Reverse Transcription ◽  
Point Of Care ◽  
Detection Methods ◽  
Clinical Samples ◽  
Complete Agreement ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Denv Serotypes

ABSTRACT The four serotypes of dengue virus (DENV) cause one of the most important and rapidly emerging mosquito-borne viral diseases in humans. Of the currently available diagnostic tests for dengue, the reverse transcription-PCR (RT-PCR) assay is the most sensitive and specific, and so it is commonly used as the gold standard. However, the requirement of a sophisticated and expensive thermal cycler makes it very difficult to use as a point-of-care diagnostic test in resource-limited regions where dengue is endemic. Tsai et al. (J Clin Microbiol 56:e01865-17, 2018, https://doi.org/10.1128/JCM.01865-17 ) report the analytical and clinical performances of a reverse transcription-insulated isothermal PCR (RT-iiPCR) assay with a portable nucleic acid analyzer for rapid detection of the four DENV serotypes; its reproducibility and complete agreement on clinical samples with the multiplex RT-PCR assay developed by the Centers for Disease Control and Prevention suggest that the dengue RT-iiPCR is a potential point-of-care test. Compared with other DENV RNA detection methods, the unique isothermal PCR design of RT-iiPCR, together with further improvements, would represent a promising new type of field-deployable diagnostic test for dengue.

scholarly journals One-Step Reverse Transcriptase PCR Method for Detection of Borrelia burgdorferi mRNA in Mouse Lyme Arthritis Tissue Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 2037-2039 ◽  
Author(s):  
F. X. Limbach ◽  
B. Jaulhac ◽  
Y. Piémont ◽  
J. L. Kuntz ◽  
H. Monteil ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Reverse Transcriptase ◽  
Simple Procedure ◽  
Lyme Arthritis ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Reverse Transcriptase Pcr ◽  
C3h Mice ◽  
One Step ◽  
Pcr Method

A one-step reverse transcriptase PCR (RT-PCR) method for detection of Borrelia burgdorferi mRNA in infected C3H mice is described. This simple procedure, less prone to nucleic acid cross-contamination than the standard method, was found to be 10-fold more sensitive than a classical two-step RT-PCR assay. By using one-step RT-PCR, flagellin mRNAs were detected in synovial and heart tissues from all seven infected mice tested.

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