Development of a rapid, cost-effective TaqMan Real-Time PCR Assay for identification and differentiation of Coccidioides immitis and Coccidioides posadasii

2009 ◽  
pp. 1-4 ◽  
Author(s):  
Kelly Sheff ◽  
Emily York ◽  
Elizabeth Driebe ◽  
Bridget Barker ◽  
Steven Rounsley ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Coccidioides Immitis ◽  
Coccidioides Posadasii ◽  
Pcr Assay

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

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.

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

2021 ◽  
Vol 15 (9) ◽  
pp. e0009765
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 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) 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 treatment and surveillance of coccidioidomycosis.

scholarly journals Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

2008 ◽  
Vol 98 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Xinshun Qu ◽  
Leslie A. Wanner ◽  
Barbara J. Christ
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Common Scab ◽  
Cost Effective ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Streptomyces Species ◽  
Standard Curve ◽  
Target Dna

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R2 = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 101 to 106 pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 103 to 106 per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

scholarly journals Development of a rapid, cost-effective TaqMan Real-Time PCR Assay for identification and differentiation ofCoccidioides immitisandCoccidioides posadasii

2010 ◽  
Vol 48 (3) ◽  
pp. 466-469 ◽  
Author(s):  
Kelly W. Sheff ◽  
Emily R. York ◽  
Elizabeth M. Driebe ◽  
Bridget M. Barker ◽  
Steven D. Rounsley ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Pcr Assay

Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

2016 ◽  
Vol 1 (1) ◽  
pp. 18
Author(s):  
Sonia Arora ◽  
Duraipandian Thavaselvam ◽  
Archna Prakash ◽  
Ashu Kumar ◽  
Anita Barua ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Cost Effective ◽  
Sybr Green ◽  
Specific Gene ◽  
Pcr Assay ◽  
Gene Target ◽  
Geographical Regions ◽  
Species Specific

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine.

2011 ◽  
Vol 29 (7_suppl) ◽  
pp. 271-271
Author(s):  
J. Millholland ◽  
S. G. Patel ◽  
C. A. Fernandez ◽  
A. P. Shuber
Keyword(s):  
Bladder Cancer ◽  
Real Time ◽  
Mutation Analysis ◽  
Real Time Pcr ◽  
Mutation Detection ◽  
Cost Effective ◽  
Optimal Performance ◽  
Single Mutation ◽  
Pcr Assay ◽  
Fgfr3 Mutations

271 Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary matrix metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine. Methods: Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. The LNA suppress wild-type DNA amplification. To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform. Results: To validate the new multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats. Conclusions: By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance. [Table: see text]

scholarly journals Quantitative real-time PCR assay for the rapid identification of the multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia

2019 ◽  
Author(s):  
Tamieka A. Fraser ◽  
Mikaela G. Bell ◽  
Patrick N.A. Harris ◽  
Scott C. Bell ◽  
Haakon Bergh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Stenotrophomonas Maltophilia ◽  
Large Scale ◽  
Direct Detection ◽  
Subsequent Development ◽  
Cost Effective ◽  
Optimal Growth ◽  
Pcr Assay ◽  
Accurate Identification

AbstractStenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia. Currently, there is no rapid, cost-effective, and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence may be underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia, with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 165 Stenotrophomonas spp. genomes identified a 4kb region specific to S. maltophilia, which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no amplification of 23 non-S. maltophilia clinical isolates. S. maltophilia was detected in 10/16 CF sputa, demonstrating the utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive, and cost-effective method for the accurate identification of S. maltophilia, and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental samples.

scholarly journals Multiplex Real-Time PCR Assay for Six Major Carbapenemase Genes

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 276
Author(s):  
Nori Yoshioka ◽  
Hideharu Hagiya ◽  
Matsuo Deguchi ◽  
Shigeto Hamaguchi ◽  
Masanori Kagita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Correlation Coefficients ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Multiplex Assay ◽  
Clinical Isolates ◽  
Pcr Assay ◽  
Detection Assay ◽  
Carbapenemase Gene

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major concern in public health. Due to the existence of the diversity of carbapenemases, development of an easily available, cost-effective multiplex detection assay for CPE is required worldwide. Using clinically available and reliable equipment, COBAS® z480 (Roche Diagnostics K.K., Tokyo, Japan), we developed a multiplex real-time PCR assay for the detection of two combinations of carbapenemases; first, blaNDM, blaKPC, and blaIMP (Set 1), and second, blaGES, blaOXA-48, and blaVIM (Set 2). We constructed standard curves for each carbapenemase gene using serial dilutions of DNA standards, then applied reference or clinical isolates with each carbapenemase gene to this assay. The multiplex assay showed satisfactory accuracy to detect CPE genes, with the correlation coefficients of greater than 0.99 for all genotypes. The assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity. Lastly, the assay successfully detected multiple genes without false-positive reactions by applying six clinical isolates carrying both NDM and OXA-48-like carbapenemase genes. Major advantages of our assay include multiplicity, simple operation, robustness, and speed (1 h). We believe that the multiplex assay potentially contributes to early diagnosis of CPE with a diverse genetic background.

scholarly journals Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Jin-Jing Geng ◽  
Zhuan-di Gong ◽  
Qyong-yi Li ◽  
Xiao-yun Shen ◽  
Suo-cheng Wei
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Minimum Detection Limit ◽  
Taqman Probe ◽  
Pcr Assay ◽  
N Protein ◽  
Qrt Pcr ◽  
Cost Effective Method

Background:  Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infection may cause losses to production by reduced weight gain, reduced milk yield. Several methods have been applied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRT-PCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technical assay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene. The main objective of the present study to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting BCoV.Materials, Methods & Results:  The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collection and processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from the known sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimized including concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, the recombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collected from diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCR were 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assay could specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL. However, universal PCR could detect only 4.72 × 103 copies/μL. Its sensitivity was 100-fold stronger than universal PCR. In conclusion, this TaqMan qRT-PCR had excellent specificity, sensitivity and stability with a 100-fold sensitivity stronger than universal PCR. Minimum detection limit was 4.72 × 101 copies/μL. This method was a cost-effective method to diagnose diarrhea and distinguish pathogens in dairy farms.Discussion:  In this study, the authors developed a quantitative real-time PCR (qRT-PCR) in this study based on the TaqMan probe of BCoV. This TaqMan qRT-PCR assay selected and used one pair of specific primers (BCoV-qF/BCoV-qR) and a specific TaqMan probe (BCoV-probe) targeting the conserved nucleocapsid (N) gene. The specificity of primers and probes was validated with Primer-BLAST. The specificity of the qRT-PCR was confirmed by the negative control and other six viruses. The findings demonstrated that TaqMan qRT-PCR could only detect BCoV. This verified the qRT-PCR had an excellent specificity. It is obvious that this TaqMan qRT-PCR assay can detect only BCoV with stronger sensitivity and reproducibility than other real-time PCR methods. The sensitivity test indicated the minimum detection limit of the TaqMan qRT-PCR was 4.72 × 101copies/μL, or 47.2 copies/μL. Sensitivity of the TaqMan qRT-PCR assay was increased by 100-fold as compared to universal PCR with a good inter-assay and intra-assay reproducibility. Thereby, based on the high sensitivity of the assay of this qRT-PCR assay it may be a cost-effective method to diagnose BCoV infections and indentify the etiologic agents of diarrhea syndrome in the dairy farms.

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