DETECTION OF CANINE PARVOVIRUS TYPE 2 BY A COMMERCIALLY AVAILABLE IN-HOUSE PCR

2020 ◽  
Vol 46 (01) ◽  
pp. 37-43
Author(s):  
Sheng-Lun Yen ◽  
Minh Hoang ◽  
Wei-Chen Wang ◽  
Ming-Lung Hung ◽  
Chi-Hsien Chien ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Canine Parvovirus ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Diagnostic Center ◽  
Low Sensitivity ◽  
Canine Parvovirus Type 2

Canine parvovirus type 2 (CPV-2) is an important pathogen that causes anorexia, vomiting, acute gastroenteritis, and bloody diarrhea. CPV-2 was divided into three variants (2a–2c) based on residue 426 of the VP2 protein. Recently, a novel Asian CPV-2c isolate was more prevalent in the Asian continent. The diagnostic tools of CPV-2 are characterized into traditional (such as immunochromatography test) and molecular methods based on their pathogen detection mechanisms. However, the low sensitivity of the immunochromatographic tests is supported by their simplicity and rapid ability to provide results for CPV-2. Surprisingly, some immunochromatography tests showed low sensitivity for the detection of novel CPV-2c compared to a real-time polymerase chain reaction (PCR) assay. Sixty rectal swabs were collected from naturally infected dogs with CPV-2 at the Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Taiwan, from 2016 to 2019. A novel in-house QubeMDx PCR system was used for diagnosis of three CPV-2 variants and compared to real-time PCR diagnosis. This diagnostic assay detected all of CPV-2 variants within 30 min and the sensitivity was identical to a real-time PCR assay using clinical samples. This study provides evidence that this novel, commercially available in-house PCR assay (QubeMDx PCR system) provides useful, simple, sensitive, reliable, and rapid diagnosis of three CPV-2 variants in veterinary clinics.

A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs

2005 ◽  
Vol 105 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Nicola Decaro ◽  
Gabriella Elia ◽  
Vito Martella ◽  
Costantina Desario ◽  
Marco Campolo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Canine Parvovirus ◽  
Pcr Assay ◽  
Canine Parvovirus Type 2

scholarly journals A SimpleProbe®real-time PCR assay for differentiating the canine parvovirus type 2 genotype

2018 ◽  
Vol 33 (1) ◽  
pp. e22654 ◽  
Author(s):  
Minh Hoang ◽  
Hung-Yi Wu ◽  
Ying-Xiu Lien ◽  
Ming-Tang Chiou ◽  
Chao-Nan Lin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Canine Parvovirus ◽  
Pcr Assay ◽  
Canine Parvovirus Type 2

scholarly journals A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus

2006 ◽  
Vol 136 (1-2) ◽  
pp. 65-70 ◽  
Author(s):  
Nicola Decaro ◽  
Gabriella Elia ◽  
Costantina Desario ◽  
Sante Roperto ◽  
Vito Martella ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Minor Groove ◽  
Canine Parvovirus ◽  
Minor Groove Binder ◽  
Pcr Assay ◽  
Minor Groove Binder Probe ◽  
A Minor

scholarly journals Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 865
Author(s):  
Germano Castelli ◽  
Federica Bruno ◽  
Stefano Reale ◽  
Simone Catanzaro ◽  
Viviana Valenza ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Parasite Load ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Predictive Values ◽  
Low Sensitivity

Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.

HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA

2012 ◽  
Vol 15 (3) ◽  
pp. 411-416 ◽  
Author(s):  
E. Osińska ◽  
A. Golke ◽  
A. Słońska ◽  
J. Cymerys ◽  
M.W. Bańbura ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Viral Dna ◽  
Herpesvirus Type ◽  
Equid Herpesvirus

Abstract Equid herpesvirus type 2 (EHV-2) together with equid herpesvirus type 5 are members of Gammaherpesvirinae subfamily, genus Rhadinovirus. EHV-2 is one of major agents causing diseases of horses common worldwide. A possible role of EHV-2 in reactivating latent equid herpesvirus type-1 has been suggested, because reactivation of latent EHV-1 was always accompanied by EHV-2 replication. Variety techniques, including cell culture, PCR and its modifications, have been used to diagnose EHV-2 infections. The aim of this study was to develop, optimize and determine specificity of real-time PCR (qPCR) for EHV-2 DNA detection using HybProbesR chemistry and to evaluate clinical samples with this method. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 70 and 7x105 copies/ml. The limit of detection (LOD) was calculated using probit analysis and was determined as 56 copies/ml. In further studies 20 different clinical samples were tested for the presence of EHV-2. Described in-house qPCR method detected viral DNA in 5 of 20 specimens used. The results of this work show that developed HybProbes-based real-time PCR assay is very reliable and valuable for detection and quantification of equid herpesvirus type 2 DNA in different clinical samples. The high level of sensitivity, accuracy and rapidity provided by the LightCycler 2.0 instrument are favorable for the use of this system in the detection of EHV-2 DNA in veterinary virology.

scholarly journals Differential Diagnosis of Fungal Pneumonias vs. Tuberculosis in AIDS Patients by Using Two New Molecular Methods

2021 ◽  
Vol 7 (5) ◽  
pp. 336
Author(s):  
Leticia Bernal-Martínez ◽  
Laura Herrera ◽  
Clara Valero ◽  
Paula de la Cruz ◽  
Larisa Ghimpu ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Histoplasma Capsulatum ◽  
Cost Effective ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Aids Patients

Opportunistic fungal pneumonias (OFP) are the main cause of death in AIDS patients worldwide. Diagnosis of these infections is often late as tuberculosis (TB) is frequently the first suspicion. In addition, diagnostic tools have limitations and are unavailable in disadvantaged regions. To perform the differential diagnosis of the main fungi causing OFP in AIDS patients (Histoplasma capsulatum, Cryptococcus neoformans/C. gattii and Pneumocystis jirovecii) vs. the Mycobacterium tuberculosis complex (MTBC), two new assays were developed: (i) a multiplex real-time PCR (MRT-PCR) and (ii) a simple and cost-effective method based on real-time PCR and the analysis of melting curves after amplification (MC-PCR). Both of the techniques were optimized and standardized “in vitro”, showing a suitable reproducibility (CV ranged between 1.84 and 3.81% and 1.41 and 4.83%, respectively), a 100% specificity and detection limits between 20 and 2 fg of genomic DNA per 20 µL of reaction. A validation study was performed by retrospectively using 42 clinical samples from 37 patients with proven fungal infection or TB, and 33 controls. The overall sensitivity for the MRT-PCR assay and the MC-PCR assay was 88% and 90.4%, respectively. Both techniques were fast, sensitive and reproducible, allowing for the detection of these pathogens and the performance of a differential diagnosis.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

2018 ◽  
Vol 56 (7) ◽  
pp. 1133-1139 ◽  
Author(s):  
Hanah Kim ◽  
Mina Hur ◽  
Eunsin Bae ◽  
Kyung-A Lee ◽  
Woo-In Lee
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Nucleic Acid Amplification ◽  
Coefficient Of Determination ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Cobas Taqman ◽  
Limit Of Quantitation ◽  
Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

scholarly journals Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 1941-1947 ◽  
Author(s):  
Alexander J. Ryncarz ◽  
James Goddard ◽  
Anna Wald ◽  
Meei-Li Huang ◽  
Bernard Roizman ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Genital Tract ◽  
Clinical Samples ◽  
Pcr Assay ◽  
The Mean ◽  
Simplex Virus

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

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