Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies

Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration ofSarcoptes scabieimites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochromecoxidase subunit 1 (cox1) gene ofS. scabiei. Thecox1gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. TheS. scabieiDNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (medianS. scabieiDNA copy number, 3.604 versus 2.457 log10copies per reaction;P= 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated.

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Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

Disease Markers ◽

10.1155/2014/836082 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8

Author(s):

Pricila da Silva Cunha ◽

Heloisa B. Pena ◽

Carla Sustek D’Angelo ◽

Celia P. Koiffmann ◽

Jill A. Rosenfeld ◽

...

Keyword(s):

Real Time ◽

Diagnostic Test ◽

Quantitative Pcr ◽

Target Genes ◽

Cost Effective ◽

Qpcr Assay ◽

Deletion Syndrome ◽

Comparative Genomic ◽

Real Time Quantitative Pcr ◽

Subtelomeric Deletion

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36:PRKCZandSKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescentin situhybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR ofPRKCZandSKIis a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

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Screening of copy number polymorphisms in human β-defensin genes using modified real-time quantitative PCR

Journal of Immunological Methods ◽

10.1016/j.jim.2005.11.001 ◽

2006 ◽

Vol 308 (1-2) ◽

pp. 231-240 ◽

Cited By ~ 24

Author(s):

QiXing Chen ◽

Malte Book ◽

XiangMing Fang ◽

Andreas Hoeft ◽

Frank Stuber

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Copy Number ◽

Real Time Quantitative Pcr

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Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01802-19 ◽

2020 ◽

Vol 58 (9) ◽

Author(s):

Megan E. Reller ◽

J. Stephen Dumler

Keyword(s):

Quantitative Pcr ◽

Scrub Typhus ◽

Spotted Fever ◽

Febrile Illness ◽

Epidemiologic Studies ◽

Qpcr Assay ◽

Spotted Fever Group ◽

Analytical Sensitivity ◽

Content Type ◽

Real Time Quantitative Pcr

ABSTRACT Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

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Applying Real-Time Quantitative PCR to Fusarium Crown Rot of Wheat

Plant Disease ◽

10.1094/pdis-91-8-1021 ◽

2007 ◽

Vol 91 (8) ◽

pp. 1021-1028 ◽

Cited By ~ 54

Author(s):

A. C. Hogg ◽

R. H. Johnston ◽

A. T. Dyer

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Field Experiments ◽

Qpcr Assay ◽

Crown Rot ◽

Yield Reduction ◽

Fusarium Crown Rot ◽

Persistent Problem ◽

Real Time Quantitative Pcr ◽

Severity Scores

Fusarium crown rot (FCR) of wheat is a persistent problem that causes significant losses worldwide. In Montana, FCR is caused primarily by Fusarium culmorum and F. pseudograminearum. Recently, a real-time quantitative PCR (QPCR) assay was developed for FCR using primers and probes specific for a segment of the trichodiene synthase (tri5) gene. The purpose of this study was to determine the utility of QPCR for accessing FCR severity on wheat in field experiments. In 2004 and 2005, plots of spring and durum wheat were inoculated with varying levels of F. pseudograminearum oat inoculum and grown under rain-fed conditions. Two weeks prior to harvest, plants were collected from the plots and assessed for FCR severity and analyzed by QPCR for Fusarium DNA quantities. Disease severity scores (DSS) and Fusarium DNA quantities were positively correlated with each other for all three cultivars in 2004 but for only the durum cultivar in 2005 (P < 0.05). In 2004, grain yields for both spring wheat cultivars were negatively correlated with Fusarium DNA quantities (P > 0.05). When DSS and Fusarium DNA quantities negatively correlated with yield, both measurements were comparable in predicting yield reduction (R = –0.64 and –0.77, respectively). Results indicate that this QPCR assay is effective in measuring FCR severity in wheat.

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Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

Applied and Environmental Microbiology ◽

10.1128/aem.06754-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 839-845 ◽

Cited By ~ 15

Author(s):

Adeline Tissier ◽

Martine Denis ◽

Philippe Hartemann ◽

Benoît Gassilloud

Keyword(s):

Drinking Water ◽

Real Time ◽

Quantitative Pcr ◽

Campylobacter Jejuni ◽

Tap Water ◽

Culture Method ◽

Cellulose Ester ◽

Campylobacter Coli ◽

Content Type ◽

Real Time Quantitative Pcr

ABSTRACTInvestigations ofCampylobacter jejuniandCampylobacter coliin samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection ofCampylobacterin 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) ofCampylobacterper 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with aC. colistrain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence ofC. jejuniandC. coligenomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination byCampylobacter.

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Quantification of Mitochondrial DNA Deletion, Depletion, and Overreplication: Application to Diagnosis

Clinical Chemistry ◽

10.1373/49.8.1309 ◽

2003 ◽

Vol 49 (8) ◽

pp. 1309-1317 ◽

Cited By ~ 45

Author(s):

Béatrice Chabi ◽

Bénédicte Mousson de Camaret ◽

Hervé Duborjal ◽

Jean-Paul Issartel ◽

Georges Stepien

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Southern Blot ◽

Quantitative Pcr ◽

Blot Analysis ◽

Copy Number ◽

Southern Blot Analysis ◽

Mtdna Copy Number ◽

Mtdna Depletion ◽

Real Time Quantitative Pcr

Abstract Background: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. Methods: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. Results: Both methods quantified 5–76% of deleted mtDNA in muscle, 59–97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r2) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. Conclusions: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.

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