Diagnostic value of a “wide-range” quantitative nested real-time PCR assay for varicella zoster virus myelitis

2013 ◽  
Vol 85 (11) ◽  
pp. 2042-2055 ◽  
Author(s):  
Teruyuki Takahashi ◽  
Masato Tamura ◽  
Toshiaki Takasu
Keyword(s):  
Varicella Zoster Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Value ◽  
Pcr Assay ◽  
Varicella Zoster ◽  
Wide Range

Development and Clinical Validation of a Multiplex Real-time PCR Assay for Herpes Simplex and Varicella Zoster Virus

2013 ◽  
Vol 22 (4) ◽  
pp. 245-248 ◽  
Author(s):  
Thean Yen Tan ◽  
Hao Zou ◽  
Danny Chee Tiong Ong ◽  
Khor Jia Ker ◽  
Martin Tze Wei Chio ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Validation ◽  
Pcr Assay ◽  
Varicella Zoster

scholarly journals Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Cyril C. Y. Yip ◽  
Siddharth Sridhar ◽  
Kit-Hang Leung ◽  
Andrew K. W. Cheng ◽  
Kwok-Hung Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Test Performance ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Varicella Zoster ◽  
Simplex Virus ◽  
Hsv 1

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

A new real-time PCR assay for detecting fungi in genus Ceratocystis

Plant Disease ◽  
2021 ◽  
Author(s):  
Karthikeyan Dharmaraj ◽  
Alice Merrall ◽  
Julie A. Pattemore ◽  
Joanne Mackie ◽  
Brett J.R Alexander ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Stem Tissue ◽  
Natural Ecosystems ◽  
Pcr Assay ◽  
Practical Applications ◽  
Wide Range

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plants species. Currently, there are over 40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostics for most Ceratocystis species currently relies on time consuming and labour-intensive culturing approaches. To provide more time efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic Taq-Man real-time PCR assay was developed using the ITS gene. This novel two-probe Taq-man assay amplified DNA from all tested Ceratocystis species. Some non-specific amplification of a few species from closely related genera was observed under certain conditions; however, these false positive detections could be ruled out using the additional PCR primers developed for further sequence based identification of the detected species. The assay was highly sensitive as it detected 0.2 pg/µl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture, enabling to detect Ceratocystis species directly from plant material, to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in case of disease outbreaks.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

2017 ◽  
Vol 80 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Aparna Tatavarthy ◽  
Laila Ali ◽  
Vikas Gill ◽  
Lijun Hu ◽  
Xiaohong Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Pcr Assay ◽  
Wide Range ◽  
The Mean ◽  
Salmonella Serotypes ◽  
Pathogen Dna ◽  
Dna Extraction Methods

ABSTRACTThe purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P < 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

Development of a Quantitative Real-Time PCR Method To Enumerate Total Bacterial Counts in Ready-to-Eat Fruits and Vegetables

2006 ◽  
Vol 69 (10) ◽  
pp. 2504-2508 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fruits And Vegetables ◽  
Bacterial Species ◽  
Culture Method ◽  
Plate Count ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Cycle Number ◽  
Wide Range

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the β subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

scholarly journals Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

2015 ◽  
Vol 53 (6) ◽  
pp. 1921-1926 ◽  
Author(s):  
Sylvie Pillet ◽  
Paul O. Verhoeven ◽  
Amélie Epercieux ◽  
Thomas Bourlet ◽  
Bruno Pozzetto
Keyword(s):  
Quality Control ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Varicella Zoster ◽  
Simplex Virus

A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.

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