Real-time PCR for mumps diagnosis on clinical specimens—Comparison with results of conventional methods of virus detection and nested PCR

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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Strip-dried blood sampling: applicability for bovine leukemia virus detection with ELISA and real-time PCR

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.11.004 ◽

2019 ◽

Vol 263 ◽

pp. 101-104 ◽

Cited By ~ 2

Author(s):

Nikolay Yu. Saushkin ◽

Jeanne V. Samsonova ◽

Alexander P. Osipov ◽

Sergey E. Kondakov

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Virus Detection ◽

Blood Sampling

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Detection and Quantification of Human Adenoviruses in Surface Waters by Nested PCR, TaqMan Real-Time PCR and Cell Culture Assays

Water Air & Soil Pollution ◽

10.1007/s11270-007-9608-5 ◽

2007 ◽

Vol 191 (1-4) ◽

pp. 83-93 ◽

Cited By ~ 27

Author(s):

M. Muscillo ◽

M. Pourshaban ◽

M. Iaconelli ◽

S. Fontana ◽

A. Di Grazia ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Nested Pcr ◽

Human Adenoviruses ◽

Detection And Quantification

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Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

Scientific Reports ◽

10.1038/s41598-021-00968-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsui-Kang Hsu ◽

Jung-Sheng Chen ◽

Hsin-Chi Tsai ◽

Chi-Wei Tao ◽

Yu-Yin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

In Silico ◽

Environmental Samples ◽

Method Development ◽

Small Volume ◽

Detection Efficiency ◽

Genotyping Method ◽

Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00505-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 3

Author(s):

Nawal El Houmami ◽

Guillaume André Durand ◽

Janek Bzdrenga ◽

Anne Darmon ◽

Philippe Minodier ◽

...

Keyword(s):

Real Time ◽

Malate Dehydrogenase ◽

Real Time Pcr ◽

Detection Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Clinical Specimens ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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

BioMed Research International ◽

10.1155/2019/5715180 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 2

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.

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Real-Time PCR Assay Compared to Nested PCR and Antigenemia Assays for Detecting Cytomegalovirus Reactivation in Adult T-Cell Leukemia-Lymphoma Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.41.9.4382-4387.2003 ◽

2003 ◽

Vol 41 (9) ◽

pp. 4382-4387 ◽

Cited By ~ 18

Author(s):

J. Ikewaki ◽

E. Ohtsuka ◽

R. Kawano ◽

M. Ogata ◽

H. Kikuchi ◽

...

Keyword(s):

T Cell ◽

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Pcr Assay ◽

Adult T Cell Leukemia ◽

Cytomegalovirus Reactivation

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Triplex Real-Time PCR without DNA Extraction for the Monitoring of Meningococcal Disease

Diagnostics ◽

10.3390/diagnostics8030058 ◽

2018 ◽

Vol 8 (3) ◽

pp. 58 ◽

Cited By ~ 1

Author(s):

Melissa Whaley ◽

Laurel Jenkins ◽

Fang Hu ◽

Alexander Chen ◽

Seydou Diarra ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Real Time ◽

Dna Extraction ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Limit Of Detection ◽

Detection Range ◽

Clinical Specimens ◽

Large Numbers ◽

Pcr Assays

Detection of Neisseria meningitidis has become less time- and resource-intensive with a monoplex direct real-time PCR (drt-PCR) to amplify genes from clinical specimens without DNA extraction. To further improve efficiency, we evaluated two triplex drt-PCR assays for the detection of meningococcal serogroups AWX and BCY. The sensitivity and specificity of the triplex assays were assessed using 228 cerebrospinal fluid (CSF) specimens from meningitis patients and compared to the monoplex for six serogroups. The lower limit of detection range for six serogroup-specific drt-PCR assays was 178–5264 CFU/mL by monoplex and 68–2221 CFU/mL by triplex. The triplex and monoplex showed 100% agreement for six serogroups and the triplex assays achieved similar sensitivity and specificity estimates as the monoplex drt-PCR assays. Our triplex method reduces the time and cost of processing CSF specimens by characterizing six serogroups with only two assays, which is particularly important for testing large numbers of specimens for N. meningitidis surveillance.

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