Real-time PCR Assay For Detection Research Articles

Evaluation of VIASURE real-time PCR assays for detection of rotavirus and norovirus GI and GII in fecal samples

Evaluation of VIASURE real-time PCR assays for detection of rotavirus and norovirus GI and GII in fecal samples

Validation of a novel real-time PCR assay for detection of HLA-B*15:02 allele for prevention of carbamazepine – Induced Stevens-Johnson syndrome/Toxic Epidermal Necrolysis in individuals of Asian ancestry

ObjectivesScreening for the HLA-B*15:02 allele has been recommended to prevent carbamazepine (CBZ) – induced Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) in individuals with Asian ancestry. We aimed, therefore, to develop and validate a robust and inexpensive method for detection of the HLA-B*15:02 allele. MethodsReal-time PCR using TaqMan® probes followed by SYBR® Green was used to detect the HLA-B*15:02 allele prior to treatment with CBZ therapy. ResultsA total of 121 samples were tested. The assay has a sensitivity of 100% (95% CI: 76.84–100.0%), a specificity of 100% (95% CI: 96.61–100%), a positive predictive value of 100% (95% CI: 76.84–100%) and a negative predictive value of 100.0% (95% CI: 96.61–100.0%), respectively. There was 100% agreement between our results and genotyping using Luminex SSO/SBT/SSP. The lowest limit of detection of the TaqMan® probe is 0.05ng/μl and the SYBR® Green is 0.5ng/μl of DNA. The unit cost of using the TaqMan® probe followed by SYBR® Green is only $4.7 USD. ConclusionWe developed a novel assay for the detection of the HLA-B*15:02 allele, which is robust, inexpensive and suitable for screening individuals of Asian ancestry in the prevention of CBZ-induced SJS/TEN.

Development of Real-time PCR Assays for Detection ofDirofilaria immitisfrom Infected Dog Blood

선형 사상충의 일종인 심장사상충은 개의 심폐 사상충증을 유발한다. 이에 본 연구의 목적은 심장사상충을 효과적으로 검출할 수 있는 실시간 중합효소연쇄반응 기법을 개발함에 있다. 연구에 있어서 사용된 프라이머 및 프로브는 선행연구에서 제작된 심장사상충 특이 프라이머 및 새롭게 제작된 TaqMan 프로브를 이용하였다. 선행연구에서 제작된 프라이머 및 농도별로 희석된 게놈유전자와 플라스미드유전자가 SYBR Green 실시간 중합효소연쇄반응 수행에 이용되었으며, 중합효소연쇄반응 과정 중 증폭 이후의 녹는 곡선의 결과를 분석하였다. 분석결과 사용된 프라이머는 각각 게놈유전자 및 플라스미드 유전자에서 특이 녹는 곡선을 나타냄에 따라 심장사상충 특이 사이토크롬 C 산화효소 유전자만을 증폭하고 있음을 확인 할 수 있었다. 새롭게 제작된 TaqMan 프로브는 SYBR Green 실시간 중합효소연쇄반응과의 결과를 농도별로 희석된 플라스미드유전자를 이용하여 비교 분석하였고, 분석결과 TaqMan 프로브를 이용한 실시간 중합효소연쇄반응이 검출효율 및 특이도에 있어서 우수함을 확인할 수 있었다. 본 연구를 통하여 개발한 실시간 중합효소연쇄반응은 기존의 전통적인 진단기법의 한계를 극복할 수 있는 신속하고 정확한 향상된 진단기법을 제시한다.

Quantitative real-time PCR assays for detection and type-specific identification of the endemic species C human adenoviruses

Human adenoviruses (HAdVs) are medically important respiratory pathogens. Among the 7 recognized species (A–G), species C HAdVs (serotypes 1, 2, 5 and 6) are globally endemic and infect most people early in life. Species C HAdV infections are most often subclinical or mild and can lead to persistent shedding from the gastrointestinal and upper respiratory tracts. They can also cause severe disseminated disease in newborn and immunocompromised persons, where rapid and quantitative detection and identification of the virus would help guide therapeutic intervention. To this end, we developed quantitative type-specific real-time PCR (qPCR) assays for HAdV-1, -2, -5 and -6 targeting the HAdV hexon gene. All type-specific qPCR assays reproducibly detected as few as 5 copies/reaction of quantified hexon recombinant plasmids with a linear dynamic range of 8 log units (5–5×107 copies). No non-specific amplifications were observed with concentrated nucleic acid from other HAdV types or other common respiratory pathogens. Of 199 previously typed HAdV field isolates and positive clinical specimens, all were detected and correctly identified to type by the qPCR assays; 10 samples had 2 HAdV types and 1 sample had 3 types identified which were confirmed by amplicon sequencing. The species C HAdV qPCR assays permit rapid, sensitive, specific and quantitative detection and identification of four recognized endemic HAdVs. Together with our previously developed qPCR assays for the epidemic respiratory HAdVs, these assays provide a convenient alternative to classical typing methods.

Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations

Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology.Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1–2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB.MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy.Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.

Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs.The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay

Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5′-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease.

Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

A TaqMan real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil

Early detection and quantification of Meloidogyne hapla in soil is essential for effective disease management. The purpose of this study was to develop a real-time PCR assay for detection of M. hapla in soil. Primers and a TaqMan probe were designed for M. hapla detection. The assay detected M. hapla and showed no significant amplification of DNA from non-target nematodes. The assay was able to detect M. hapla in a background of plant and soil DNA. A dilution series of M. hapla eggs in soil showed a high correlation (, ) with Ct values. The assay could predict root-knot development in carrots by testing soils before planting. The assay could be useful for management decisions in carrot cultivation.

Nested Real-Time PCR法を用いたカキからのノロウイルス検出の有効性

Nested Real-Time PCR法を用いたカキからのノロウイルス検出の有効性

Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human β-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1457-x) contains supplementary material, which is available to authorized users.

A real-time PCR assay for detection of the Ehrlichia muris-like agent, a newly recognized pathogen of humans in the upper Midwestern United States

The Ehrlichia muris-like agent (EMLA) is an emerging, tick-transmitted human pathogen that occurs in the upper Midwestern United States. Here, we describe the development and validation of a p13-based quantitative real-time PCR TaqMan assay to detect EMLA in blood or tissues of ticks, humans, and rodents. The primer and probe specificities of the assay were ascertained using a large panel of various Ehrlichia species and other members of Rickettsiales. In addition to control DNA, both non-infected and EMLA-infected human blood, Mus musculus blood, and M. musculus tissue extracts were evaluated, as were non-infected and EMLA-infected Ixodes scapularis and uninfected Dermacentor variabilis DNA lysates. The specificity of the probe was determined via real-time PCR. An EMLA p13 control plasmid was constructed, and serial dilutions were used to determine the analytical sensitivity, which was found to be 1 copy per 4μl of template DNA. The sensitivity and specificity of this assay provides a powerful tool for ecological studies involving arthropod vectors and their mammalian hosts.

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

BackgroundThe porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1.ResultsA pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 100 ~ 109 copies/μL. This method doesn’t detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5 %. The serum virus titers of P1 positive in this study were measured by this protocol to be 103 to 107 copies/mL.ConclusionsThe established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.

Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis

Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains of E. coli that can cause bloody diarrhea and kidney failure. Seven STEC serogroups, O157, O26, O45, O103, O111, O121 and O145 are responsible for more than 71% of the total infections caused by this group of pathogens. All seven serogroups are currently considered as adulterants in non-intact beef products in the U.S. In this study, two multiplex melt curve real-time PCR assays with internal amplification controls (IACs) were standardized for the detection of eight STEC serogroups. The first multiplex assay targeted E. coli serogroups O145, O121, O104, and O157; while the second set detected E. coli serogroups O26, O45, O103 and O111. The applicability of the assays was tested using 11 different meat and produce samples. For food samples spiked with a cocktail of four STEC serogroups with a combined count of 10CFU/25g food, all targets of the multiplex assays were detected after an enrichment period of 6h. The assays also worked efficiently when 325g of food samples were spiked with 10CFU of STECs. The assays are not dependent on fluorescent-labeled probes or immunomagnetic beads, and can be used for the detection of eight STEC serogroups in less than 11h. Routine preliminary screening of STECs in food samples is performed by testing for the presence of STEC virulence genes. The assays developed in this study can be useful as a first- or second-tier test for the identification of the eight O serogroup-specific genes in suspected food samples.

Clinical evaluation of a novel Fast-track diagnostics multiplex real-time PCR assay for detection and quantification of Hepatitis E virus

Clinical evaluation of a novel Fast-track diagnostics multiplex real-time PCR assay for detection and quantification of Hepatitis E virus

New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group.

Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm(41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm(41), inactive erm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.

Development and evaluation of a real-time PCR assay for detection of lobster, a crustacean shellfish allergen

Crustacean shellfish are a leading cause of food allergy in American adults, and the Food Allergen Labeling and Consumer Protection Act requires that different types of crustacean shellfish be distinguished from each other. In general ELISA assays are not capable of differentiating crustacean type, but PCR assays are. In this work, a real-time PCR assay for lobster, a crustacean shellfish allergen, was developed and evaluated. Food matrices were spiked with lobster meat at 0.1, 1, 10, 100, 1000, 104, and 105 parts per million (ppm). In addition to testing of several different food matrices, method performance was determined using conditions which have historically proven challenging for PCR analyses, specifically food matrices with low DNA content and acidic pH levels, as well as foods that were treated with combined high temperature and pressure. Real-time PCR standard curves were generated from spiked, treated foods and analyzed with respect to linear range and reaction efficiency. In most cases, the assay was linear over 6–8 orders of magnitude; lower limits of detection were 0.1–1 ppm and reaction efficiencies were within the preferred range of 100 ± 10%. A notable exception occurred in the case of heat treatment at acidic pH, which resulted in severe delay or complete loss of amplification signals.

A real-time PCR assay for detection and quantification of Botrytis cinerea in Pelargonium x hortorum plants, and its use for evaluation of plant resistance

A reliable EvaGreen-based qPCR assay was developed for the relative quantification of fungal and plant DNAs in the Botrytis cinerea-Pelargonium × hortorum pathosystem. Primers, designed on RPB2 (DNA-dependent RNA polymerase subunit II) gene sequences directed the amplification of a specific 149 bp product for B. cinerea. No cross-reactivity was observed in DNA extracted from several nontarget fungi and bacterial, and in pelargonium plants, with the exception of the closely related species Botryotinia pelargonii and Botrytis fabae. This assay allowed for quantification of as little as 1.55 pg fungal DNA and 9.95 pg plant DNA, and detection of the pathogen in both symptomatic and asymptomatic pelargonium plants. The qPCR protocol is suitable for studying cultivar resistance and induced resistance in pelargonium plants, which requires accurate quantification of the pathogen in diseased host tissue. This assay allowed us to establish that the Pelargonium × hortorum cvs. ‘XL Boomerang’ and ‘Eroica 2000’ showed high foliar resistance to B. cinerea with respect to the susceptible cv. ‘Fernando’, and that spray applications of 0.3 mM acibenzolar-S methyl markedly protect pelargonium plants against gray mold. The protection for plants treated with chitosan (0.2 %) did not reach significance.

Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia.

Aspergillus fumigatus causes varied clinical syndromes ranging from colonization to deep infections. The mainstay of therapy of Aspergillus diseases is triazoles but several studies globally highlighted variable prevalence of triazole resistance, which hampers the management of aspergillosis. We studied the prevalence of resistance in clinical A. fumigatus isolates during 4 years in a referral Chest Hospital in Delhi, India and reviewed the scenario in Asia and the Middle East. Aspergillus species (n = 2117) were screened with selective plates for azole resistance. The isolates included 45.4% A. flavus, followed by 32.4% A. fumigatus, 15.6% Aspergillus species and 6.6% A. terreus. Azole resistance was found in only 12 (1.7%) A. fumigatus isolates. These triazole resistant A. fumigatus (TRAF) isolates were subjected to (a) calmodulin and β tubulin gene sequencing (b) in vitro antifungal susceptibility testing against triazoles using CLSI M38-A2 (c) sequencing of cyp51A gene and real-time PCR assay for detection of mutations and (d) microsatellite typing of the resistant isolates. TRAF harbored TR34/L98H mutation in 10 (83.3%) isolates with a pan-azole resistant phenotype. Among the remaining two TRAF isolates, one had G54E and the other had three non-synonymous point mutations. The majority of patients were diagnosed as invasive aspergillosis followed by allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. The Indian TR34/L98H isolates had a unique genotype and were distinct from the Chinese, Middle East, and European TR34/L98H strains. This resistance mechanism has been linked to the use of fungicide azoles in agricultural practices in Europe as it has been mainly reported from azole naïve patients. Reports published from Asia demonstrate the same environmental resistance mechanism in A. fumigatus isolates from two highly populated countries in Asia, i.e., China and India and also from the neighboring Middle East.

Collaborative trial validation of cry1Ab/Ac and Pubi-cry TaqMan-based real-time PCR assays for detection of DNA derived from genetically modified Bt plant products

Presence of genetic modifications in rice products originating from China and imported to the European Union market is detected since 2006. Neither these products from China nor any other genetically modified rice lines are approved as food or feed in the EU. The transgenic rice varieties identified contain genetic elements and constructs coding for insect-resistance genes from Bacillus thuringiensis (Bt) coding for insecticidal crystal (cry) proteins. In particular, DNA sequences coding for codon-optimised or fused cry1Ab/c genes and constructs driven by the maize ubiquitin promoter (P-ubiZM1) were identified. For improved screening and identification of genetic modifications present in Asian rice products, two TaqMan-based real-time PCR assays targeting codon-optimised cry1Ab/Ac and the Pubi-cry construct have been developed. These assays have been validated in an international collaborative trial with 17 participants from 10 countries. Based on a new mathematical–statistical model and an adjusted experimental set-up of the collaborative trial, a close examination of the limit of detection (LOD95%) and the probability of detection of the qualitative PCR assays was conducted. The evaluation of the method performance characteristics and results of the collaborative trial validation are presented.