scholarly journals Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

2021 ◽  
Vol 11 ◽  
Author(s):  
Suhua Xin ◽  
Hong Zhu ◽  
Chenglin Tao ◽  
Beibei Zhang ◽  
Lan Yao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Epidemiologic Study ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Chicken Farm

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

scholarly journals A Novel Real-Time PCR Assay for the Rapid Detection of Virulent Streptococcus equi Subspecies zooepidemicus—An Emerging Pathogen of Swine

2021 ◽  
Vol 8 ◽  
Author(s):  
Suresh V. Kuchipudi ◽  
Meera Surendran Nair ◽  
Michele Yon ◽  
Abhinay Gontu ◽  
Ruth H. Nissly ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
High Mortality ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Streptococcus Equi ◽  
Swine Herds ◽  
Pcr Targeting

Streptococcus equi subspecies zooepidemicus, a zoonotic bacterial pathogen caused a series of outbreaks with high mortality affecting swine herds in multiple locations of the USA and Canada in 2019. Further genetic analysis revealed that this agent clustered with ATCC 35246, a S. zooepidemicus strain associated with high mortality outbreaks in swine herds of China originally reported in 1977. Rapid and accurate diagnosis is absolutely critical for controlling and limiting further spread of this emerging disease of swine. Currently available diagnostic methods including bacteriological examination and PCR assays do not distinguish between the virulent strains and avirulent commensal strains of S. zooepidemicus, which is critical given that this pathogen is a normal inhabitant of the swine respiratory tract. Based on comparative analyses of whole genome sequences of the virulent isolates and avirulent sequences, we identified a region in the SzM gene that is highly conserved and restricted to virulent S. zooepidemicus strains. We developed and validated a novel probe-based real-time PCR targeting the conserved region of SzM. The assay was highly sensitive and specific to the virulent swine isolates of Streptococcus equi subspecies zooepidemicus. No cross reactivity was observed with avirulent S. zooepidemicus isolates as well as other streptococcal species and a panel of porcine respiratory bacterial and viral pathogens. The PCR efficiency of the assay was 96.64 % and was able to detect as little as 20 fg of the bacterial DNA. We then validated the diagnostic sensitivity and specificity of the new PCR assay using a panel of clinical samples (n = 57) and found that the assay has 100% sensitivity and specificity as compared to bacteriological culture method. In summary, the PCR assay will be an extremely valuable tool for the rapid accurate detection of virulent swine S. zooepidemicus isolates and directly from clinical samples.

Soil Baiting, Rapid PCR Assay and Quantitative Real Time PCR to Diagnose Late Blight of Potato in Quarantine Programs

2018 ◽  
Vol 4 ◽  
pp. 25-32 ◽  
Author(s):  
Touseef Hussain ◽  
Bir Pal Singh ◽  
Firoz Anwar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Pcr Assay ◽  
Diagnostic Laboratory ◽  
Rapid Pcr ◽  
Accurate Detection

Phytophthora infestans (mont) de Bary is a pathogen of great concern across the globe, and accurate detection is an important component in responding to the outbreaks of potential disease. Although the molecular diagnostic protocol used in regulatory programs has been evaluated but till date methods implying direct comparison has rarely used. In this study, a known area soil samples from potato fields where light blight appear every year (both A1 and A2 mating type) was assayed by soil bait method, PCR assay detection and quantification of the inoculums. Suspected disease symptoms appeared on bait tubers were further confirmed by rapid PCR, inoculums were quantified through Real Time PCR, which confirms presence of P. infestans. These diagnostic methods can be highly correlated with one another. Potato tuber baiting increased the sensitivity of the assay compared with direct extraction of DNA from tuber and soil samples. Our study determines diagnostic sensitivity and specificity of the assays to determine the performance of each method. Overall, molecular techniques based on different types of PCR amplification and Real-time PCR can lead to high throughput, faster and more accurate detection method which can be used in quarantine programmes in potato industry and diagnostic laboratory.

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 Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

2015 ◽  
Vol 53 (12) ◽  
pp. 3935-3937 ◽  
Author(s):  
Daniel Golparian ◽  
Stina Boräng ◽  
Martin Sundqvist ◽  
Magnus Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Content Type ◽  
Dna Assay ◽  
Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

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 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.

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.

Roles of the multiplex real-time PCR assay and β-D-glucan in a high-risk population for intra-abdominal candidiasis (IAC)

2019 ◽  
Vol 58 (6) ◽  
pp. 789-796 ◽  
Author(s):  
J Fortún ◽  
M J Buitrago ◽  
F Gioia ◽  
E Gómez-Gª de la Pedrosa ◽  
M E Alvarez ◽  
...  
Keyword(s):  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Volume Overload ◽  
Diagnostic Methods ◽  
Practice Volume ◽  
High Risk Population ◽  
Pcr Assay ◽  
European Consensus ◽  
Abdominal Candidiasis

Abstract Multiplex quantitative real-time PCR (MRT-PCR) using blood can improve the diagnosis of intra-abdominal candidiasis (IAC). We prospectively studied 39 patients with suspected IAC in the absence of previous antifungal therapy. Blood cultures, MRT-PCR, and β-D-glucan (BDG) in serum were performed in all patients. IAC was defined according to the 2013 European Consensus criteria. For MRT-PCR, the probes targeted the ITS1 or ITS2 regions of ribosomal DNA. Candidaemia was confirmed only in four patients (10%), and IAC criteria were present in 17 patients (43.6%). The sensitivity of MRT-PCR was 25% but increased to 63.6% (P = .06) in plasma obtained prior to volume overload and transfusion; specificity was above 85% in all cases. BDG performance was improved using a cutoff &gt; 260 pg/ml, and improvement was not observed in samples obtained before transfusion. In this cohort of high risk of IAC and low rate of bloodstream infection, the performance of non-culture-based methods (MRT-PCR or BDG) was moderate but may be a complementary tool given the limitations of diagnostic methods available in clinical practice. Volume overload requirements, in combination with other factors, decrease the accuracy of MRT-PCR in patients with IAC.

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