Development of a Highly Sensitive Genus-Specific Quantitative Reverse Transcriptase Real-Time PCR Assay for Detection and Quantitation of Plasmodium by Amplifying RNA and DNA of the 18S rRNA Genes

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

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Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay

PLoS ONE ◽

10.1371/journal.pone.0227143 ◽

2020 ◽

Vol 15 (1) ◽

pp. e0227143

Author(s):

Angela Nagel ◽

Emmanouela Dimitrakopoulou ◽

Norbert Teig ◽

Peter Kern ◽

Thomas Lücke ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Universal Screening ◽

Cmv Infection ◽

Pcr Assay ◽

Screening Approach ◽

Highly Sensitive ◽

Congenital Cmv Infection ◽

Congenital Cmv

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A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽

10.1128/msphere.00658-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Christian Shema Mugisha ◽

Hung R. Vuong ◽

Maritza Puray-Chavez ◽

Adam L. Bailey ◽

Julie M. Fox ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Real Time Pcr ◽

Rna Extraction ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Dna Viruses ◽

Culture Supernatants ◽

Hiv 1 ◽

Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

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Dasytricha Dominance in Surti Buffalo Rumen Revealed by 18S rRNA Sequences and Real-Time PCR Assay

Current Microbiology ◽

10.1007/s00284-011-9975-4 ◽

2011 ◽

Vol 63 (3) ◽

pp. 281-288 ◽

Cited By ~ 8

Author(s):

K. M. Singh ◽

A. K. Tripathi ◽

P. R. Pandya ◽

D. N. Rank ◽

R. K. Kothari ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Pcr Assay ◽

Surti Buffalo ◽

Rrna Sequences ◽

Buffalo Rumen

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Real-time PCRs for detection of Trichomonas vaginalis β-tubulin and 18S rRNA genes in female genital specimens

Journal of Medical Microbiology ◽

10.1099/jmm.0.47163-0 ◽

2007 ◽

Vol 56 (6) ◽

pp. 772-777 ◽

Cited By ~ 27

Author(s):

Paul Simpson ◽

Geoff Higgins ◽

Ming Qiao ◽

Russell Waddell ◽

Tuckweng Kok

Keyword(s):

Real Time ◽

Trichomonas Vaginalis ◽

18S Rrna ◽

Clinical Manifestations ◽

Developed Countries ◽

Rrna Genes ◽

Rrna Gene ◽

Wet Mount ◽

Increased Risk ◽

18S Rrna Genes

Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. The incidence of trichomoniasis in developed countries has decreased substantially during the past decade, but high prevalence of this disease can still be found in rural and remote areas of Australia. Clinical manifestations of symptomatic women are generally non-specific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. Current diagnosis of T. vaginalis relies on the visualization of motile organisms in a wet-mount preparation. Culture is used mainly in reference laboratories. The latter two methods require viable organisms and would not be suitable for use where transportation of specimens can be delayed. Two real-time fluorescence resonance energy transfer (FRET) hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the β-tubulin and 18S rRNA genes. We tested 500 randomly selected female patients, in an STD setting, for T. vaginalis DNA. The FRET PCRs targeting the β-tubulin gene and the 18S rRNA gene detected 96 % (85/89) and 100 % (89/89) , respectively, of the positive specimens (first-void urine sample or genital swabs). Wet-mount microscopy was performed on 76 of these PCR-positive specimens and showed a sensitivity of 38 % (29/76). The prevalence, by PCR, of trichomoniasis was 18 % in this study. The two real-time PCRs developed in this study, targeting different genetic regions of the organism, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.

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Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.026781-0 ◽

2011 ◽

Vol 60 (4) ◽

pp. 459-466 ◽

Cited By ~ 29

Author(s):

Spencer D. Polley ◽

Samuel Boadi ◽

Julie Watson ◽

Alan Curry ◽

Peter L. Chiodini

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Enterocytozoon Bieneusi ◽

Sybr Green ◽

Pcr Assay ◽

Real Time Analysis ◽

Highly Sensitive ◽

Stool Samples

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

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Development of a duplex real-time PCR assay for the differentiation of Blastomyces dermatitidis and B. gilchristii and a retrospective study of blastomycosis in New York (2005-2019)

10.1101/2020.08.11.20172932 ◽

2020 ◽

Author(s):

Mitchell Kaplan ◽

YanChun Zhu ◽

Julianne V Kus ◽

Lisa McTaggart ◽

Vishnu Chaturvedi ◽

...

Keyword(s):

New York ◽

Retrospective Study ◽

North America ◽

Real Time ◽

Real Time Pcr ◽

Blastomyces Dermatitidis ◽

Pcr Assay ◽

Rare Finding ◽

Clinical Specimens ◽

Highly Sensitive

Blastomycosis due to Blastomyces dermatitidis and B. gilchristii is a notable cause of respiratory mycoses in North America with recurrent outbreaks. We developed a highly sensitive, specific, and reproducible Taqman duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii. The new assay permitted retrospective analysis of Blastomyces cultures (2005 to 2019), and primary clinical specimens (2013-2019) from NY patients. Blastomyces dermatitidis was the causal agent for the majority of 38 cases while B. gilchristii was implicated in five cases; a rare finding reported from New York. The duplex real-time PCR assay will be useful for further understanding of ecology and epidemiology of blastomycosis caused by B. dermatitidis and B. gilchristii.

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Evaluating Performance of Multiplex Real Time PCR for the Diagnosis of Malaria at Elimination Targeted Low Transmission Settings of Ethiopia.

10.21203/rs.3.rs-572301/v1 ◽

2021 ◽

Author(s):

Mahlet Belachew ◽

Mistire Wolde ◽

Desalegn Nega ◽

Bokretsion Gidey ◽

Legessie Negash ◽

...

Keyword(s):

Light Microscopy ◽

Real Time ◽

Malaria Elimination ◽

Real Time Pcr ◽

Nucleic Acid Amplification ◽

Molecular Testing ◽

Pcr Assay ◽

Blood Samples ◽

Low Transmission ◽

Highly Sensitive

Abstract Background: Malaria incidence has declined in Ethiopia in the past ten years. Current malaria diagnostic tests, including light microscopy and antigen-detecting rapid tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. Thus, this study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. Methods: A health facility based cross sectional survey was conducted in selected malaria sentinel sites. Malaria suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 were enrolled into this study. Socio demographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood sample (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at EPHI malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other.Results: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as P. falciparum infection, 16 as P. vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as Pf, 18 as PV and RDT detected 43 as Pf, 17 as PV, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI [93-100]) and 83.2% (95% CI [77.6-87.9]), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI [46.9-68.4] and 67% (95% CI [56.2-76.7]); and 100% (95% CI [98-100] and 98.9 (95% CI (96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. Conclusions: Multiplex real time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination program, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

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