Real-Time PCR Assay for Detection of a New Simulant for Poxvirus Biothreat Agents

ABSTRACT Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant “simulant” microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the Cydia pomonella granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per μl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows Cydia pomonella granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies.

Download Full-text

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-1133 ◽

2018 ◽

Vol 56 (7) ◽

pp. 1133-1139 ◽

Cited By ~ 7

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.

Download Full-text

Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Foods ◽

10.3390/foods9030332 ◽

2020 ◽

Vol 9 (3) ◽

pp. 332

Author(s):

Jasmin Wrage ◽

Oxana Kleyner ◽

Sascha Rohn ◽

Jürgen Kuballa

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Detection Method ◽

Immunoglobulin E ◽

Limit Of Detection ◽

Cocos Nucifera ◽

Cross Reactivity ◽

Pcr Assay ◽

Probe System ◽

Ige Mediated

So far, only a few cases of immunoglobulin E (IgE)-mediated coconut allergies have been described in the literature. Due to a growing consumption of coconut-containing foods in occidental countries, the number of coconut allergies may also increase. As there is no causative immunotherapy in clinical routine, appropriate food labelling is particularly important, also with regard to cross-contamination, to prevent serious health consequences. The purpose of this study was to develop a DNA-based detection method for coconut (Cocos nucifera). Initially, three sets of coconut-specific primers were designed and tested. A TaqMan™ probe was then developed to identify and quantify coconut by real-time PCR assay. With 27 other plant and animal species, the specificity of the primer/probe system was tested and cross reactivity was excluded. In a dilution series, a limit of detection of 1 pg/µL was determined. Thus, the developed real-time PCR assay is a suitable method to detect coconut in food.

Download Full-text

Real-Time PCR Detection of Parapoxvirus DNA,

Clinical Chemistry ◽

10.1373/clinchem.2005.060335 ◽

2006 ◽

Vol 52 (2) ◽

pp. 316-319 ◽

Cited By ~ 53

Author(s):

Andreas Nitsche ◽

Mathias Büttner ◽

Sonja Wilhelm ◽

Georg Pauli ◽

Hermann Meyer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Probit Analysis ◽

Animal Origin ◽

Pcr Assay ◽

Medical Practitioners ◽

B2l Gene ◽

Zoonotic Infections

Abstract Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.

Download Full-text

Evaluation of a novel real-time PCR assay for the detection, identification and quantification of Plasmodium species causing malaria in humans

Malaria Journal ◽

10.1186/s12936-021-03842-8 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Kim van Bergen ◽

Toon Stuitje ◽

Robert Akkers ◽

Eric Vermeer ◽

Rob Castel ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

False Negative ◽

Performance Characteristics ◽

Analytical Performance ◽

Melting Curve Analysis ◽

Mixed Infections ◽

Plasmodium Ovale ◽

Pcr Assay

Abstract Background The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value. Methods The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions. Results No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10–3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%. Conclusion Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.

Download Full-text

A novel and sensitive real-time PCR system for universal detection of poxviruses

Scientific Reports ◽

10.1038/s41598-021-81376-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Léa Luciani ◽

Lucia Inchauste ◽

Olivier Ferraris ◽

Rémi Charrel ◽

Antoine Nougairède ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Virus Discovery ◽

Pcr Assay ◽

Potential Health ◽

Smallpox Virus ◽

Health Threat ◽

Poxvirus Genome ◽

Universal Detection

AbstractSuccess in smallpox eradication was enabled by the absence of non-human reservoir for smallpox virus. However, other poxviruses with a wider host spectrum can infect humans and represent a potential health threat to humans, highlighted by a progressively increasing number of infections by (re)emerging poxviruses, requiring new improved diagnostic and epidemiological tools. We describe here a real-time PCR assay targeting a highly conserved region of the poxvirus genome, thus allowing a pan-Poxvirus detection (Chordopoxvirinae and Entomopoxvirinae). This system is specific (99.8% for vertebrate samples and 99.7% for arthropods samples), sensitive (100% for vertebrate samples and 86.3% for arthropods samples) and presents low limit of detection (< 1000 DNA copies/reaction). In addition, this system could be also valuable for virus discovery and epidemiological projects.

Download Full-text

A rapid and automated sample-to-result Candida auris real-time PCR assay for high-throughput testing of surveillance samples with BD MAX™ open system

10.1101/608190 ◽

2019 ◽

Author(s):

L. Leach ◽

A. Russell ◽

Y. Zhu ◽

S. Chaturvedi ◽

V. Chaturvedi

Keyword(s):

New York ◽

Real Time ◽

High Throughput ◽

Open System ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Pcr Assay ◽

Candida Auris ◽

Clinical Sensitivity

ABSTRACTThe multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX™ open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX™ optimum assay conditions were: DNA extraction at 75°C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX™ System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX™ assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.

Download Full-text

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

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.609126 ◽

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%.

Download Full-text

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

10.21203/rs.3.rs-267291/v1 ◽

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.

Download Full-text

Development of a real-time PCR assay and comparison to CHROMagarTM STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa

African Journal of Laboratory Medicine ◽

10.4102/ajlm.v6i1.609 ◽

2017 ◽

Vol 6 (1) ◽

Cited By ~ 3

Author(s):

John B. Kalule ◽

Karen H. Keddy ◽

Anthony Smith ◽

Mark P. Nicol ◽

Lourens Robberts

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Predictive Value ◽

Limit Of Detection ◽

Reference Standard ◽

Pcr Assay ◽

Diagnostic Technology ◽

Sensitivity Specificity

Introduction: Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates.Objectives: This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples.Methods: This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates.Results: The real-time PCR limit of detection was 5.3 target copies/μL of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%.Conclusions: The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.

Download Full-text

Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

10.1101/2021.04.08.21254780 ◽

2021 ◽

Author(s):

Sudha Chaturvedi ◽

Tanya R Victor ◽

Anuradha Marathe ◽

Ketevan Sidamonidze ◽

Kelly L Crucillo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Cross Reactivity ◽

Coccidioides Immitis ◽

Coccidioides Posadasii ◽

Pcr Assay ◽

Valley Fever ◽

Clinical Specimens ◽

Wide Range

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 129 isolates and three primary specimens as C. posadasii and 23 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the surveillance of coccidioidomycosis.

Download Full-text