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 Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

2015 ◽  
Vol 53 (7) ◽  
pp. 2042-2048 ◽  
Author(s):  
S. W. Peterson ◽  
I. Martin ◽  
W. Demczuk ◽  
A. Bharat ◽  
L. Hoang ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Dna Sequence ◽  
Real Time Pcr ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Specific Marker ◽  
Molecular Assay ◽  
Pcr Assay ◽  
Content Type

The incidence of antimicrobial-resistantNeisseria gonorrhoeaecontinues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) inponA,mtrR,penA,porB, and oneN. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24N. gonorrhoeae-negative NAAT specimens, and 34N. gonorrhoeae-positive NAAT specimens. Twenty-four of theN. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252N. gonorrhoeaestrains, the agreement between the DNA sequence and real-time PCR was 100% forporA,ponA, andpenA, 99.6% formtrR, and 95.2% forporB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% forporB, 95.8% forponAandmtrR, and 91.7% forpenA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins inN. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

scholarly journals A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry

2021 ◽  
Vol 8 ◽  
Author(s):  
Suresh V. Kuchipudi ◽  
Michele Yon ◽  
Meera Surendran Nair ◽  
Maurice Byukusenge ◽  
Rhiannon M. Barry ◽  
...  
Keyword(s):  
Real Time ◽  
Respiratory Disease ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Isolation And Identification ◽  
Pcr Efficiency ◽  
Bacteriological Diagnosis ◽  
Avibacterium Paragallinarum

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.

scholarly journals Establishment and Application of a Taqman Reverse Transcriptase Quantitative Real Time Pcr Assay for Feline Calicivirus

2021 ◽  
Author(s):  
jingjie zhao ◽  
Lin Liang ◽  
Guangzhi Zhang ◽  
Wenhui Li ◽  
Shaohan Li ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Feline Calicivirus ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Virus Content ◽  
Nasal Swabs

Abstract Feline calicivirus (FCV) is an infectious pathogen that causes disease in cats. With the current emergence of FCV-associated virulent systemic disease (FCV VSD) worldwide, the establishment of a rapid, sensitive, and reproducible diagnostic assay for its detection is important to inform prevention and control strategies. In this study, specific primers and TaqMan-FAM probes were designed based on the conserved regions of the FCV genome sequence, and a TaqMan reverse transcriptase quantitative real time PCR assay was established. This assay could specifically detected the FCV genome. The assay had a wide dynamic range, with linear detection in the range of 9.6×109 copies/μL to 9.6×100 copies/μL, with a limit of detection of 9.6×100 copies/μL, showing high sensitivity and repeatability. In addition, we used this assay to evaluated clinical samples (n=100) taken from cats from across China for the presence/absence of FCV genetic material For samples with low virus content, the positive detection rate of TaqMan reverse transcriptase quantitative real time PCR assay (RT-qPCR) was much higher than that of conventional reverse transcriptase PCR assay (cRT-PCR). And The qRT-PCR assay was used to detect the viral load of cat swabs within 17 days after FCV infection. From days 1-9, the oral and nasal swabs generally had higher viral loads than the anal swabs. While from days 10-17, the levels in the oral and nasal swabs being generally lower than those in the anal swabs. Overall, this FCV TaqMan RT-qPCR assay assay represents a rapid and accurate.

scholarly journals Simultaneous detection and differentiation of clinically relevant relapsing fever Borrelia with semi-multiplex real-time PCR

2021 ◽  
Author(s):  
Elizabeth A. Dietrich ◽  
Adam J. Replogle ◽  
Sarah W. Sheldon ◽  
Jeannine M. Petersen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Hard Tick ◽  
Emerging Pathogens ◽  
Pcr Assay ◽  
Relapsing Fever ◽  
Multiplex Pcr Assay

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semi-multiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (B. hermsii and others), the emerging hard tick transmitted pathogen B. miyamotoi, and the agent of louse-borne relapsing fever (B. recurrentis). The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae, and multiple TaqMan probes to allow for detection of and differentiation among the three groups. The assay detects all RF borreliae tested with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single assay real-time PCR approach will help to improve diagnosis of RF by simplifying the selection of tests to aid in clinical management of acutely ill RF patients.

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 Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

2020 ◽  
Author(s):  
zhenhua Guo ◽  
Kunpeng Li ◽  
Songlin Qiao ◽  
Xinxin Chen ◽  
Ruiguang Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Limit Of Detection ◽  
African Swine Fever ◽  
Economic Losses ◽  
Clinical Samples ◽  
Diagnosis Method ◽  
Pcr Method ◽  
And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

scholarly journals Direct detection of microorganisms responsible for the sexually transmitted infection on the mobile real-time PCR device without treating in advance

2021 ◽  
Author(s):  
Masaaki Muraoka ◽  
Kazunori Sohma ◽  
Osamu Kawaguchi ◽  
Mikio Mizukoshi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Treponema Pallidum ◽  
Nucleic Acid Amplification ◽  
Direct Pcr ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Ct Value

ABSTRACTAs WHO reported, four curable STIs-chlamydia, gonorrhoea, syphilis and trichomoniasis occur more than 1 million per each day globally almond 2016. For this reason, it is important to control these STIs, one of which is “to detect”. The general methods in order to detect STIs are nucleic acid amplification tests (NAATs). One of the reasons why NAATs are utilized in many tests is that it is possibly to be more sensitive than other test. However, there needs to treat extraction of nucleic acids in advance and amplify specific regions by NAATs, and hence it must take much labour and much time. In this work, for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP) which is each etiological agent of chlamydia, gonorrhoea and syphilis, we evaluate and propose “quicker and simpler” NAATs. Specifically, utilizing mobile real-time PCR device “PCR1100” and PCR reagent kit “KAPA3G Plant PCR Kit”, it was considered whether real-time direct PCR could be performed or not without treating DNA extraction in advance so-called “direct”.As a result, firstly, we established that real-time direct PCR could be performed in all of CT, NG, and TP, and moreover, each Ct value correlated with the concentration of each organism similarly to detection of genome DNA (each correlation coefficient R2 > 0.95). Moreover, each assay demonstrated a limit of detection (LOD) of the follows; CT was 10^0.86 = 7.24 IFU/reaction, NG was 10^-0.19 = 0.65 CFU/reaction, and TP was 10^1.4 = 25.1 organisms/reaction. However, it appeared the sensitivity was a little low, especially for CT and TP.Secondly, we found that even as without treating sample in advance, the time of detection was required more less 15 minutes at any of case, which was very quick compared with other current methods for real-time PCR. Additionally, compared with other commercial devices, it was easier to operate the PCR1100 device, for example, start, analysis of Ct value.In conclusion, the present study has demonstrated that it is possible for real-time direct PCR to perform with combination of the PCR1100 device and the PCR reagent kit in 3 kinds of microorganisms-CT, NG and TP. Furthermore, we propose “quicker and simpler” methods for NAATs, which it would not take labour and time. Further studies are needed in order to contribute to control STIs.

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.

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