Rapid Real-time PCR Assay Research Articles

Rapid and accurate detection of fish allergen within 30 min using a food nucleic acid releaser combined with rapid real time PCR assay

To achieve rapid on-site nucleic acid detection of fish allergens in food, this study established a new direct and rapid qPCR detection technology for fish. The technology was able to complete nucleic acid extraction in 4 min using a food nucleic acid releaser, and the DNA samples were amplified by rapid qPCR within 25 min. The results showed that direct-rapid qPCR can specifically detect fish, and it can also detect 0.00001 % of fish components in artificially simulated samples, with high detection precision. This method can be used to effectively detect fish in various deep-processed foods. As a simple, rapid, and accurate molecular detection method for fish allergens, this method has broad application prospects for on-site food safety detection.

Development of a Rapid and High-Throughput Multiplex Real-Time PCR Assay for Mycoplasma hominis and Ureaplasma Species

Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, and may cause nongonococcal urethritis, pelvic inflammatory disease, and infertility. Mycoplasma hominis and Ureaplasma species may also cause severe invasive infections in immunocompromised patients. Current culture-based methods for Mycoplasma/Ureaplasma identification are costly and laborious, with a turnaround time between 1 and 2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma species in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historic culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percentage agreement and 100% negative percentage agreement) and 96% for Ureaplasma species (96% positive percentage agreement and 97% negative percentage agreement). Specimen stability was validated for up to 7 days at room temperature. This multiplex molecular assay was designed for implementation in a high-complexity clinical microbiology laboratory. With this method, >90 samples can be tested in one run, with a turnaround time of 4 to 5 hours from specimen extraction to reporting of results. This PCR test is also more labor effective and cheaper than the conventional culture-based test, thus improving laboratory efficiency and alleviating labor shortages.

Rapid and scalable DNA extraction and real-time PCR assay from boxwood tissue for the detection of Calonectria pseudonaviculata, causal agent of boxwood blight.

Boxwood blight can be challenging to detect in the field, especially when symptoms are mild, thus requiring large numbers of plants to be screened. Therefore, a rapid diagnostic assay that can detect the pathogen from large amounts of plant tissue would be useful. Here, we present a crude DNA extraction protocol that is rapid and scalable. The DNA extraction protocol can process large volumes of tough boxwood tissue rapidly without using cetyltrimethylammonium bromide (CTAB) or phenol-chloroform to remove inhibitors. Additionally, to detect the boxwood blight pathogen, Calonectria pseudonaviculata, we developed a TaqMan probe to use with previously described PCR primers for a real-time PCR assay. The assay's limit of detection was determined by diluting symptomatic boxwood leaves in non-diseased leaves and by adding spores to non-diseased leaves to simulate diagnostic scenarios. The assay was able to detect the pathogen in symptomatic leaves diluted up to 1×104 to 1×105 fold in non-diseased leaves, and from as low as 1,000 to 10,000 spores added to 1.2 g of non-diseased leaves. The ability to extract DNA from large volumes of plant tissue facilitates screening more plant tissue using the real-time PCR assay without increasing the number of samples to process in the lab.

Retrospective and Comparative Study of Three Molecular Assays for the Macrolide Resistance Detection in Mycoplasma genitalium Positive Urogenital Specimens.

The capacity of Mycoplasma genitalium to develop resistance to macrolides makes detection of macrolide resistance genes by rapid real-time PCR assays increasingly necessary in clinical diagnostic laboratories so as to initiate appropriate treatment as rapidly as possible. The aim of this retrospective and comparative study was to conduct the clinical evaluation of three commercially available kits for macrolide resistance detection. A total of 111 M. genitalium positive samples analyzed in the Clinical Microbiology Laboratory of the Miguel Servet University Hospital, Zaragoza (Spain) were used. After M. genitalium molecular confirmation, the three assays under study were evaluated and discrepant results were resolved via sequencing. The clinical sensitivity for resistance detection was 83% (95% confidence interval, 69% to 93%) for the ResistancePlus® MG panel kit (SpeeDx Pty Ltd., Sydney, Australia), 95% (84% to 99%) for AllplexTM MG & AziR Assay (Seegene®, Seoul, Korea), and 97% (88% to 99%) for the VIASURE macrolide resistance-associated mutations (23SrRNA) Real time PCR detection kit (Certest Biotec, Zaragoza, Spain). The clinical specificity was 100% (94% to 100%) for Allplex and VIASURE assays and 95% (86% to 99%) for SpeeDx assay. The results arising from this study are cause for strong consideration for the implementation of rapid real-time PCR assays in clinical diagnosis laboratories to eliminate treatment failure and transmission as soon as possible.

Development of a direct duplex real-time PCR assay for rapid detection of domestic cat hepadnavirus.

Domestic cat hepadnavirus (DCH) is a novel hepadnavirus, first identified in 2018. DCH is generally detected using conventional PCR assays, which include time-consuming agarose gel electrophoresis. We developed a rapid, sensitive, and specific real-time PCR (rtPCR) assay for the detection of the DCH genome. To streamline the procedure, our rtPCR assay was carried out using blood samples, without DNA extraction. A consensus primers/probe set was designed based on the nucleotide sequences of the surface/polymerase gene of all DCH strains available in GenBank. To exclude the possibility that the PCR reaction was blocked by anticoagulants, we also used a primers/probe set for amplifying the cat beta-actin gene as a reference gene. Our direct duplex rtPCR assay had high sensitivity, with a limit of detection of 10 copies/μL of blood for DCH. Our direct duplex rtPCR assay should be a useful tool for DCH detection and surveillance.

Molecular detection of Escherichia coli isolated from human, animal and environmental samples targeting mdh gene: A simple one health approach

Background: Escherichia coli cause a wide range of infections in humans and animals in hospitals and community associated with multidrug-resistance, and serves as bioindicator of fecal contamination as well as AMR. Aim: Early detection of this bioindicator from humans, animals and environment facilitates characterization with respect to AMR and bacterial forensics for effective public health and infection control measurements. Method: We investigated a simple and rapid species-specific end-point and real-time PCR assays targeting malate dehydrogenase (mdh) gene to detect Escherichia coli isolated from livestock, human, and environmental water in Midnapore, West Bengal, India in a timely and cost-effective approach. Results: Of 126 E. coli isolates, 53, 41 and 32 were cultured from livestock, human and environmental water. The mdh gene was present in 120 (95.2%) isolates, including all environmental isolates. Two (3.8%) animal and four human (9.8%) isolates were discordant between culture and PCR results. The PCR assays produced results in about 90 min with a running cost of US$3-5 per sample. Conclusion: Both the PCR assays may be utilized in detection of Escherichia coli depending on the laboratory infrastructure.

Development of a rapid and sensitive reverse transcription real-time quantitative PCR assay for detection and quantification of grass carp reovirus II

Hemorrhagic disease of grass carp, which is induced by grass carp reovirus II (GCRV-II), leads to mass mortality in grass carp culture and causes enormous economic loss. However, there is currently no quantitative analysis method for the detection of GCRV-II, which is greatly restricted the etiological and epidemiological study of the disease. In this study a reverse transcription TaqMan PCR (RT-qPCR) assay was developed for the quantitative detection of GCRV-II. The probe and primers targeted location is the segment 6 (S6) region of the GCRV-II genome which is highly conserved. Standard curves were drawn and criteria were confirmed after the determination of the optimum reaction conditions. The species-specific assay showed that the method is highly specific and has no cross reactions with other pathogens. The assay was sufficiently sensitive to detect as low as 10 copies of virus RNA. Moreover, the method has a very good repeatability for batches and inter-batches sample detection. Then the method was applied to detect the virus in tissue samples from clinically infected grass carp, compared with conventional RT-seminested PCR, the RT-qPCR represents a specific value for detection rate of positive samples. In summary, the RT-qPCR was applied and achieved high sensitivity and specificity for GCRV-II detection.

Establishment of a duplex TaqMan RT-PCR for the differential detection of RHDV GI.1 and GI.2

BackgroundRabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal hepatitis of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). Up to 2010, all RHD viruses (RHDV) isolated belonged to one genotype. In 2010, a new genotype of RHDV (RHDV2/b, currently designated GI.2 based on phylogenetic analysis) emerged in France. The aim of this study was to develop a rapid, simple, specific and sensitive TaqMan real-time PCR assay for the classic strain of RHDV and RHDV2 detection. Specific primers and probes were designed for the VP60 gene of RHDV and RHDV2 within the conserved region of viral genome. ResultsThis study was demonstrated to be highly specific for RHDV and RHDV2, without cross-reactions with other non-targeted viruses. The detection limit of this work was 102 copies of RHDV and RHDV2, respectively. The coefficient of variation of the assay was less than 5% for both intra-assay and inter-assay. The reproducibility of method was assessed using plasmids and the coefficient of variation obtained was 0.2–3.70. Of 79 clinical samples, 68 were positive samples (86.08%), of which 60 were classic RHDV variants (75.9%), 4 were co-infected (5.06%) and 8 were RHDV2 (10.12%), those results are more sensitivity compare with conventional RT-PCR RT-PCR. ConclusionsIn conclusion, this duplex TaqMan RT-qPCR based on VP60 gene of RHDV and RHDV2 could be a valuable tool in diagnose and molecular epidemiological study of the RHDV and RHDV2.

Use of real-time PCR as an alternative to conventional genotyping methods for the laboratory detection of lymphogranuloma venereum (LGV)

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method.

Molecular Epidemiology of Third-Generation-Cephalosporin-Resistant Enterobacteriaceae in Southeast Queensland, Australia.

Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. In addition to routine mass spectrometry and antibiotic sensitivity testing, isolates were examined using rapid (∼40-min) real-time PCR assays targeting the most common extended-spectrum β-lactamases (ESBLs; blaCTX-M-1 and blaCTX-M-9 groups, plus blaTEM, blaSHV, and an internal 16S rRNA gene control). AmpC CMY β-lactamase (blaCMY) prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes, and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one β-lactamase gene, with 144 (50%) harboring blaCTX-M-1 group, 92 (31.9%) harboring blaCTX-M-9 group, 48 (16.7%) harboring blaSHV, 133 (46.2%) harboring blaTEM, and 34 (11.8%) harboring blaCMY genes. A subset of isolates (n = 98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants and to verify genotyping accuracy. WGS of β-lactamase-negative or carbapenem-resistant isolates identified uncommon ESBL and carbapenemase genes, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples

Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-μL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention–approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.

A real-time PCR assay based on a specific mutation of PstS1 gene for detection of M. bovis strains

The Mycobacterium tuberculosis complex (MTBC) is composed of several genetically related and pathogenic mycobacterial species, including M. tuberculosis, M. bovis and M.africanum et al. In our previous study, we found that M. bovis strains had a unique SNP located in position 1055 in the sequence of the pstS1 gene in which a T was substituted by a C. In this study, specific primers and MGB probes were designed according to the mutation in PstS1 gene, and a sensitive, specific and rapid real-time PCR assay for M. bovis was established. Then the assay was used to detect M. bovis in simulation samples. The minimum detectable concentration is 101 copies for M. bovis DNA. The standard curve showed correlation coefficient between threshold cycle and PstS1 gene fragment copy number was 0.997 and slope is −3.144. The minimum detectable concentration is 101 cells/ml for simulation sample. In addition, M.bovis strain 93006, 14 clinical BCG stains and 7 clinical M.bovis strain showed positive while the other strains showed negative results, which proved good specificity. This assay had high sensitivity and specificity for identification of M. bovis from the simulation specimens. The assay can be applied for epidemiological and ecological surveillance of M. bovis strains.

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3

The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/μL and 61.2 copies/μL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.

Frequency of Influenza-A-H1N1 in Patients with Community-Acquired Pneumonia Admitted to Loghman Hakim Hospital

Background: Here we assessed the incidence of Influenza-A-H1N1-related pneumonia in community-acquired pneumonia (CAP) at Loghman Hakim Hospital, Tehran, Iran. M aterials and Methods: In this prospective study from November 22, 2016, to June 21, 2017, patients with CAP and suspected to seasonal influenza were included. Rapid Antigen test and quantitative real-time PCR assay were performed on samples. P-value < 0.05 was considered significant. In addition, radiologic patterns of them were evaluated. R es ults: a total of 29 admitted CAP patients were suspected of seasonal influenza. Two cases out of them were positive for influenza by real-time PCR, similar to result of influenza rapid test. The most common finding in their chest X ray was consolidation in one lobe. None of them vaccinated against influenza. Only nine patients received empiric Oseltamivir treatment. The amount of irrational antibiotic administration was significant. C onclusion: Despite low statistical numbers, admitted influenza CAP patients did not have unusual symptoms and radiologic patterns. Other results in this study showed need for antibiotic stewardship program and better training about necessity of vaccination.

Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.

Development of a SYBR Green I real-time PCR for detection and quantitation of orthopoxvirus by using Ectromelia virus

Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed. This assay was able to detect a minimum of 10 copies of standard DNA and 5 TCID50 units of ECTV. In addition, no cross-reactions were observed with two DNA viruses, such as herpes simplex virus and swine pseudorabies virus, and one RNA virus, vesicular stomatitis virus. Furthermore, intra- and inter-assay variability data showed that this method had a highly reproducibility and reliability. Moreover, the current assay was faster and had a higher sensitivity for detection of ECTV genomic DNA in cell cultured and clinical test samples. Therefore, the high sensitivity and reproducibility of this SYBR Green real-time PCR approach is a more effective method than the conventional PCR for ECTV diagnosis and quantitation.

Corrigendum.

Sayler KA, Bigelow T, Koster LG, Swenson S, Bounds C, Hernández F, Wisely SM. Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds. J Vet Diagn Invest 2017;29:522-528. (Original doi:10.1177/1040638717706593). In the article titled "Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds" by Katherine A. Sayler et al., the Acknowledgements section, should read as follows: We thank all of the researchers who provided isolates or DNA samples of PRV, including C Romero at the University of Florida, College of Veterinary Medicine (U.S.); A Moreno at the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (Italy); S Verpoest at the CODA-CERVA Veterinary and Agrochemical Research Center (Belgium); A Steinrigl at the Austrian Agency for Health and Food Safety GmbH, Institute for Veterinary Disease Control (Austria); Z Dirbakova at the State Veterinary and Food Institute, Veterinary Institute in Zvolen (Slovakia); T Müller at the Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health (Germany); and F Ruiz-Fons and D González-Barrio at the Instituto de Investigación en Recursos Cinegéticos (IREC) (Spain).

Development of a rapid and reliable TaqMan probe-based real-time PCR assay for the detection and enumeration of the multifaceted yeast Kluyveromyces marxianus in dairy products

Kluyveromyces marxianus is a multifaceted yeast used in the dairy, biotechnological, and probiotic industries, and has been implicated in the spoilage of dairy products. Currently, K. marxianus detection involves laborious and time-consuming culture methods. To develop a rapid and reliable detection method for K. marxianus in pure cultures and dairy products, we designed a novel real-time PCR primer/probe set, KM55, which targets the internal transcribed spacer (ITS) rRNA gene. In the inclusivity and exclusivity tests, 11 K. marxianus strains tested positive, while 28 closely related microorganisms tested negative; this confirmed 100% sensitivity and specificity of the assay. Further, the KM55 set enabled a linear K. marxianus detection in the range of 100–105 colony-forming units (CFU) per reaction in phosphate-buffered saline, 10% skim milk, and commercial yogurts, with no cross-reactivity with microorganisms of kefir origin. The enumeration of K. marxianus in kefir revealed 5.1 to 6.9 logCFU ml−1 of kefir, depending on the fermentation time, temperature, and grain-milk ratio. K. marxianus was successfully detected in artificially contaminated yogurt samples within 2 h. These results suggest that this method could be an effective and sensitive presumptive screening tool for detecting and enumerating K. marxianus in dairy products.

Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3

Porcine circovirus 3 (PCV3) is a novel circovirus that was associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation. The objective of this study was to develop a rapid, simple, specific and sensitive TaqMan-based real-time PCR assay for PCV3 detection. Specific primers and probe were designed for the cap gene of PCV3 within the conserved region of viral genome. The assay was highly specific for PCV3, without cross-reactions with other non-targeted pig viruses. The detection limit of this assay was 102 copies. The assay had an efficiency of 95.7%, a regression squared value (R2) of 0.994 and showed a linear range of 102–107 copies PCV3 DNA per reaction. The assay was also very reproducible, with the intra- and inter-assay coefficient of variation less than 2.0%. For the 112 archived clinical samples collected from 2014 to March 2017, the PCV3 positive ratio was 12.5% (14/112) with the real-time PCR. The presence of the PCV3 dated back to at least 2014 in China and samples collected in 2017 had the highest PCV3 positive ratio (46.7%, 7/15). The real-time PCR assay could be used for detection of PCV3 in epidemiological and pathogenesis studies.