scholarly journals Four-color multiplex real-time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Olivier Thellin ◽  
Benaïssa Elmoualij ◽  
Willy Zorzi ◽  
Jorgen S. Jensen ◽  
Renaud Close ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Plasmid Dna ◽  
Limit Of Detection ◽  
Analytical Data ◽  
Mycoplasma Genitalium ◽  
Clinical Samples ◽  
Resistance Mutations ◽  
Pcr Assay ◽  
Azithromycin Resistance

Abstract Background The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient’s antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. Methods Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. Results The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. Conclusions A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.

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 Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Lei Ma ◽  
Fanwen Zeng ◽  
Bihong Huang ◽  
Feng Cong ◽  
Ren Huang ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Watery Diarrhea ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Molecules ◽  
Positive Rate

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

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 Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays

2022 ◽  
Vol 9 (1) ◽  
pp. 1-20
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Respiratory Pathogens ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

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.

scholarly journals 149. Extraction-free RT-PCR to Detect SARS-CoV-2 Variants of Concern

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S89-S91
Author(s):  
Brian L Harry ◽  
Yue Qiu ◽  
Ling Lu ◽  
Mara Couto-Rodriguez ◽  
Dorottya Nagy-Szakal ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Research Support ◽  
Pcr Assay ◽  
Synthetic Controls ◽  
Novel Coronavirus

Abstract Background SARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. Methods The CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. Results Similar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1a-mut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Detection of VOC in clinical specimens and validation by NGS Conclusion The combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing. Disclosures Brian L. Harry, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Shi-Long Lu, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder)

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