Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

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Single-copy detection of somatic variants from solid and liquid biopsy

Scientific Reports ◽

10.1038/s41598-021-85545-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ana-Luisa Silva ◽

Paulina Klaudyna Powalowska ◽

Magdalena Stolarek ◽

Eleanor Ruth Gray ◽

Rebecca Natalie Palmer ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Real Time Pcr ◽

Clinical Decision Making ◽

High Sensitivity ◽

Clinical Decision ◽

Single Copy ◽

Turnaround Time ◽

Copy Detection ◽

Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

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Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽

10.3390/toxins12120757 ◽

2020 ◽

Vol 12 (12) ◽

pp. 757

Author(s):

Sara Franco Ortega ◽

Ilenia Siciliano ◽

Simona Prencipe ◽

Maria Lodovica Gullino ◽

Davide Spadaro

Keyword(s):

Food Safety ◽

Real Time ◽

Aspergillus Flavus ◽

Real Time Pcr ◽

Limit Of Detection ◽

Screening Method ◽

Lamp Assay ◽

High Sensitivity ◽

Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

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Real-time evaluation of an optimized real-time PCR assay versus Brilliance chromogenic MRSA agar for the detection of meticillin-resistant Staphylococcus aureus from clinical specimens

Journal of Medical Microbiology ◽

10.1099/jmm.0.025288-0 ◽

2011 ◽

Vol 60 (3) ◽

pp. 323-328 ◽

Cited By ~ 12

Author(s):

J. Danial ◽

M. Noel ◽

K. E. Templeton ◽

F. Cameron ◽

F. Mathewson ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Real Time ◽

Real Time Pcr ◽

Test Performance ◽

High Sensitivity ◽

Turnaround Time ◽

Homology Search ◽

Pcr Assay ◽

Indirect Measure ◽

The Mean

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

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Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Microbiology Insights ◽

10.4137/mbi.s38517 ◽

2016 ◽

Vol 9 ◽

pp. MBI.S38517 ◽

Cited By ~ 15

Author(s):

Jing Zhang ◽

Guo-Chiuan Hung ◽

Kenjiro Nagamine ◽

Bingjie Li ◽

Shien Tsai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Candida Species ◽

Rapid Detection ◽

Corneal Transplantation ◽

High Sensitivity ◽

Turnaround Time ◽

Pcr Assays ◽

Primer Sets ◽

Pcr Primer

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

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Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites

PLoS ONE ◽

10.1371/journal.pone.0252887 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252887

Author(s):

Renate Schneider ◽

Aline Lamien-Meda ◽

Herbert Auer ◽

Ursula Wiedermann-Schmidt ◽

Peter L. Chiodini ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Melting Curve ◽

Resonance Energy ◽

High Sensitivity ◽

Significant Rise ◽

Quantitative Detection ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

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Detection and Quantification of Milk Ingredients as Hidden Allergens in Meat Products by a Novel Specific Real-Time PCR Method

Biomolecules ◽

10.3390/biom9120804 ◽

2019 ◽

Vol 9 (12) ◽

pp. 804 ◽

Cited By ~ 4

Author(s):

Caterina Villa ◽

Joana Costa ◽

Isabel Mafra

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Meat Products ◽

Milk Proteins ◽

12S Rrna ◽

Processed Meat ◽

Rrna Gene ◽

Wide Range ◽

Detection And Quantification

Milk ingredients are often included in a wide range of meat products, such as cooked hams and sausages, to improve technological characteristics. However, milk proteins are also important food allergens. The aim of this study was the development of a highly sensitive and specific real-time PCR system targeting the 12S rRNA gene of Bos domesticus for the detection and quantification of milk as an allergenic ingredient in processed meat products. The method was able to achieve an absolute limit of detection (LOD) of 6 fg of milk DNA. Using a normalized approach (∆Ct method) for the detection of milk protein concentrate (MPC), it was possible to obtain sensitivities down to 0.01% (w/w) of MPC in model hams (raw and cooked) and autoclaved sausages, and 0.005% in raw sausage mixtures. The developed systems generally presented acceptable PCR performance parameters, being successfully validated with blind samples, applied to commercial samples, and further compared with an immunochemical assay. Trace amounts of milk material were quantified in two out of 13 samples, but the results mostly infer the excessive practice of the precautionary labeling.

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Assessment of real-time PCR for diagnosis ofMycoplasma pneumoniaepneumonia in pediatric patients

Canadian Journal of Microbiology ◽

10.1139/w05-118 ◽

2006 ◽

Vol 52 (2) ◽

pp. 125-129 ◽

Cited By ~ 23

Author(s):

Miyuki Morozumi ◽

Akira Ito ◽

Somay Y Murayama ◽

Keiko Hasegawa ◽

Reiko Kobayashi ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Mycoplasma Pneumoniae ◽

Real Time Pcr ◽

High Sensitivity ◽

Community Acquired Pneumonia ◽

Clinical Samples ◽

Rrna Gene ◽

The 16S Rrna Gene

We developed a real-time PCR to detect Mycoplasma pneumoniae with a primer set designed for the 16S rRNA gene. Clinical samples (n = 937) were collected from children with community-acquired pneumonia between April 2002 and March 2004 at 12 Japanese medical institutions. Sensitivity of real-time PCR was calculated as 10 colony-forming units per reaction tube using a pMP01 plasmid carrying a 225-bp target DNA fragment of the 16S rRNA gene in M. pneumoniae M129, a standard strain. Results, obtained within 2 h, were compared with those of conventional culture and serologic methods. Of all cases tested, 151 (16.4%) and 129 (13.8%) were positive for M. pneumoniae by real-time PCR and by culture, respectively. Among the 151 cases, almost all of those tested serologically by passive agglutination showed a rise in M. pneumoniae antibody titre between acute and convalescent sera. We conclude that this real-time PCR can identify M. pneumoniae rapidly and fulfills the need for rapid identification, high sensitivity, and high specificity.Key words: real-time PCR, Mycoplasma pneumoniae identification, pediatrics, community-acquired pneumonia, Mycoplasma pneumoniae culture.

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Identification of Selected Tuna Species in Commercial Products

Molecules ◽

10.3390/molecules26041137 ◽

2021 ◽

Vol 26 (4) ◽

pp. 1137

Author(s):

Eliska Servusova ◽

Zora Piskata

Keyword(s):

Real Time ◽

Muscle Tissue ◽

Internal Control ◽

Real Time Pcr ◽

Limit Of Detection ◽

Fish Muscle ◽

Coefficient Of Determination ◽

12S Rrna ◽

Rrna Gene ◽

Additional Species

This study was conducted to develop systems for the identification of four tuna species (skipjack tuna Katsuwonus pelamis, yellowfin tuna Thunnus albacares, bullet tuna Auxis sp. and Atlantic bonito Sarda sp). At first, raw samples of these species and a mix intended as internal control were prepared for the authentication of fish muscle tissue of the genus Thunnus sp., Auxis sp. and Sarda sp. DNA from raw muscle tissue, the mix and samples was extracted with the DNeasy mericon Food Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of DNA in raw samples were evaluated using a spectrophotometer. Primers and probe sequences were specifically designed to identify the selected species. In addition, primers and a probe for the endogenous 12S rRNA gene were designed to determine the presence of amplifiable fish (especially tuna) DNA in samples. Furthermore, the species specificity of the designed primers and probes was verified in DNA samples of various tuna and bonito species. Limit of detection for the selected species was calculated as well as the coefficient of determination R2 and efficiency of real-time PCR testing was determined. To evaluate the developed real-time PCR methods, 70 commercial tuna products were analysed. The results show that mislabelling of fish products can still be encountered and, moreover, the presence of an additional species can be identified.

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Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.00623-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6570-6578 ◽

Cited By ~ 22

Author(s):

Sonia E. Létant ◽

Gloria A. Murphy ◽

Teneile M. Alfaro ◽

Julie R. Avila ◽

Staci R. Kane ◽

...

Keyword(s):

Bacillus Anthracis ◽

Real Time ◽

Incubation Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Limit Of Detection ◽

Turnaround Time ◽

Pcr Analysis ◽

Content Type ◽

Pcr Method

ABSTRACTIn the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulentBacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for theB. anthracischromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulentB. anthracisin environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

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Optimization and validation of a quadruplex real-time PCR assay for the diagnosis of diphtheria

10.1101/600270 ◽

2019 ◽

Author(s):

Edgar Badell ◽

Sophie Guillot ◽

Marie Tulliez ◽

Marine Pascal ◽

Leonardo-Gabriel Panunzi ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Clinical Samples ◽

Rrna Gene ◽

Gene Target ◽

Corynebacterium Ulcerans ◽

Toxigenic Strains ◽

Processing Control ◽

Roche Diagnostics

AbstractDiphtheria is caused by toxigenic strains ofCorynebacterium diphtheriae, Corynebacterium ulceransandCorynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysaet al. J Med Microbiol. 2016 65(12):1521-1527). Here, we present an improvement of the quadruplex method, in which a 16S rRNA gene target was added as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays. This improved qPCR method was validated using 36 bacterial isolates and 16 clinical samples. The method allows detection of thetoxgene and distinguishingC. diphtheriae(including the newly described speciesC. belfantii) fromC. ulceransandC. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness of the method to temperature and reagent concentration variations were demonstrated. The lower limit of detection forC. diphtheriae, C. ulceransandtoxtargets was 1.86 genome copies per 5 μL reaction volume. Finally, the method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England – National Infection Service, London, UK). This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

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