Competitive Internal Amplification Control Research Articles

Design and Optimization of a yst-PCR to Detect Yersinia enterocolitica in Meat Food.

In this study, a polymerase chain reaction (PCR) directed to the yst chromosomal gene (yst-PCR) was used as a rapid, sensitive, and specific method to detect Yersinia enterocolitica strains belonging to different biotypes in foods; a competitive Internal Amplification Control (cIAC) is also developed. The cIAC had a molecular weight of 417 bp and was detected until a concentration of 0.85 ng/μL. No other strains of other Yersinia species, nor Enterobacteriales order were detected by this PCR. In pure culture, the detection limit (DL) of the yst-PCR was lower for ystA+ strain (10 colony-forming unit [CFU]/mL) than for ystB+ strain (1 × 102 CFU/mL); which was the concentration detected in Y. enterocolitica inoculated minced meat. The proposed protocol included an enrichment step in peptone sorbitol bile (PSB) broth at 25°C for 24 h followed by isolation on Mac Conkey agar and chromogenic medium. An aliquot of the PSB broth homogenate and a loopful from the confluent zone of solid media were collected to perform DNA extraction for yst-PCR, and typical colonies were characterized by biochemical assays. Among 30 non-contaminated food samples, 4 samples were yst-positive and no Y. enterocolitica isolates were obtained. It is suggested that this yst-PCR could be used in the investigation of Y. enterocolitica in foods.

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

Isothermal Amplification with a Target-Mimicking Internal Control and Quantitative Lateral Flow Readout for Rapid HIV Viral Load Testing in Low-Resource Settings.

Point-of-care diagnostics often use isothermal nucleic acid amplification for qualitative detection of pathogens in low-resource healthcare settings but lack sufficient precision for quantitative applications such as HIV viral load monitoring. Although viral load (VL) monitoring is an essential component of HIV treatment, commercially available tests rely on relatively high-resource chemistries like real-time polymerase chain reaction and are thus used on an infrequent basis for millions of people living with HIV in low-income countries. To address the constraints of low-resource settings on nucleic acid quantification, we describe a recombinase polymerase amplification and lateral flow detection approach that quantifies HIV-1 DNA or RNA by comparison to a competitive internal amplification control (IAC) of a known copy number, which may be set to any useful threshold (in our case, a clinically relevant threshold for HIV treatment failure). The IAC is designed to amplify alongside the HIV target with a similar efficiency, allowing for normalization of the assay to variation or inhibition and enabling an endpoint readout that is compatible with commercially available kits for nucleic acid lateral flow detection and interpretable with minimal instrumentation or by the naked eye. We find that this approach can reliably differentiate ≤600 or ≥1400 copies of HIV DNA from a 1000-copy threshold when lateral flow strips are imaged with a conventional office scanner and analyzed with free densitometry software. We further demonstrate a user-friendly adaptation of this analysis to process cell phone photographs with an automated script. Alternatively, we show via a survey that 21 minimally trained volunteers could reliably resolve ≥10-fold (log10) differences of HIV DNA or RNA by naked eye interpretation of lateral flow results. This amplification and detection workflow requires minimal instrumentation, takes just 30 min to complete, and when combined with a suitable sample preparation method, may enable HIV VL testing while the patient waits or a self-test, which has the potential to improve care. This approach may be adapted for other applications that require quantitative analysis of a nucleic acid target in low-resource settings.

Development and evaluation of multiplex PCR for detection of T-2 and zearalenone producing Fusarium spp.

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2×103 CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2×104 CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.

A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease.

Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting M. haemolytica/M. bovis and P. multocida/H. somni, respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes (tetH, tetR, msrE, mphE, sul2, floR, erm42), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.

Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control.

Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of E. coli O157:H7, enabling accurate quantification and preventing false negative results in E. coli O157:H7 contaminated samples.

A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling

ObjectivesPertussis is a highly transmissible acute respiratory infection caused by the bacterial pathogen Bordetella pertussis. The purpose of this study was to develop a rapid, simple and sensitive diagnostic test for detecting this pathogen. MethodsHere we present a recombinase aided amplification (RAA) assay incorporating competitive internal amplification control (IAC) to detect Bordetella pertussis using the DNA obtained by boiling. This assay was performed in a single closed tube at 39°C within 30min. A total of 115 clinical samples suspected of pertussis were collected and tested by the internally controlled RAA assay using both extracted DNA with the commercial kit and the DNA obtained by boiling. For comparison, the real-time PCR (RT-PCR) was also performed with DNA extraction in parallel. ResultsThe sensitivity of the internally controlled RAA assay was 101 copies or 10CFU/ml per reaction in detecting plasmid DNA or B. pertussis strain. The optimum concentration of the IAC plasmid was determined to be 100 copies, and the introduction of IAC effectively reduced the occurrence of false negatives. Compared to the RT-PCR, RAA results with DNA extraction obtained 100% sensitivity and specificity, and the RAA results with heat-treated DNA showed 85.96% sensitivity and 100% specificity. ConclusionWith the advantages of 45min turn-around time and simple steps of DNA purification, this assay could become a useful diagnostic tool for Bordetella pertussis detection and is potentially suitable for point-of-care identification to guide prompt clinical treatment.

A Hexaplex PCR Assay Developed for Simultaneous Detection of Bacillus anthracis and Yersinia pestis and Distinguish Their Virulence Levels Tested on Vietnamese Samples

Bacillus anthracis and Yersinia pestis are at high risk of bioterrorism, but they have different strains of virulence that exist in the natural environment, making it difficult to diagnose the risk of disease. This study aimed to develop a novel hexaplex PCR assay to simultaneously detect B. anthracis and Y. pestis and quickly distinguish their virulence levels. The vrrA, pagA, and capA genes from B. anthracis and ypo2088, pla, and caf1 genes from Y. pestis were specifically amplified to generate six PCR fragments of 222, 751, and 610 and 103, 438, and 271 bp, respectively. Furthermore, a recombinant competitive internal amplification control was designed in the assay, which coamplified with the capA gene primer pairs, producing a fragment of 1000 bp in length. Thirty-eight bacterial strains including 4 reference strains and 34 screening strains derived from human, herbivores, and environments in the North of Vietnam, as well as spiked soil samples, were used to evaluate the assay. The new hexaplex PCR assay was in accordance with the conventional culture methods to detect B. anthracis and Y. pestis. In addition, this assay can be quickly discriminated among pathogenic and non-pathogenic B. anthracis and Y. pestis strains, as well as rapidly differentiate levels of virulence among strains isolated from human and soil samples in some areas of Vietnam. The new hexaplex PCR is a useful tool in screening for both pathogenic and non-pathogenic B. anthracis and Y. pestis, necessary in use for biodefense purposes and disease prevention.

Targeting Other Mycotoxin Biosynthetic Genes.

Real-time PCR (qPCR) methods are adequate tools for sensitive and rapid detection and quantification of toxigenic molds contaminating food commodities. Methods of qPCR for quantifying zearalenone (ZEA)-, sterigmatocystin (ST)-, cyclopiazonic acid (CPA)-, and patulin (PAT)-producing molds have been designed on the basis of specific target genes involved in the biosynthesis of these mycotoxins. In this chapter reliable qPCR protocols to detect and quantify such toxigenic molds are described. All of these methods are suitable when working with mold pure cultures and mold contaminated foods. For ZEA-producing molds, two qPCR using the SYBR Green fluorochrome and based on two polyketide synthase (PKS) genes are detailed. qPCR protocols relied on the fluG and the idh genes able to quantify ST- and PAT-producing molds, respectively, which can be performed by both SYBR Green and TaqMan methodologies are described. Regarding CPA-producing molds a TaqManq PCR method including a competitive internal amplification control is detailed. Since DNA extraction is a critical step in the detection and quantification of toxigenic molds by qPCR, a protocol for extracting DNA from mold pure cultures and food is also described.

Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces

A conventional PCR targeted directly to the detection of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stools of symptomatic patients may require the introduction of internal controls to detect false negative results. In the present study, we designed a competitive internal amplification control (IAC) to be included in a well-known PCR protocol used to amplify the stx1and stx2 genes from STEC isolates. The IAC was introduced in the PCR reaction and amplified when E. coli O157:H7 cultures and contaminated pediatric feces were assayed. When STEC concentration was 103 CFU ml−1 in pure culture and 104 CFU g−1 in contaminated stools, the IAC at concentration of 0.143 pg μl−1 in the PCR reaction mixture was co-amplified with the stx2 sequence, producing bands of 279 and 349 bp, respectively. These STEC values were considered the detection limits of the duplex PCR. The specific detection of STEC by duplex PCR including IAC might be achieved directly on pediatric feces when the pathogen load reaches concentrations of at least 104 CFU g−1.

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation.

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

Designing novel and simple competitive internal amplification control for reliable PCR diagnosis of herpes simplex virus.

Background:PCR is a molecular technique for herpes simplex virus (HSV) detection that can cause life-threatening infections such as encephalitis and keratitis. However, the main issues, false-negative results causing by PCR inhibitors, of this technique that reduce PCR efficiency. To overcome this problem, a competitive internal amplification control (IAC) was constructed for conventional PCR using the PCR-cloning technique.Objectives:The purpose of this study is the design of competitive IAC for PCR diagnosis of HSV, which in fact is the main cause of keratitis and viral encephalitis in developed countries.Materials and Methods:Composite primers for PCR amplification of Leishmania major kDNA (kinetoplast DNA) were designed and optimized to use as IAC-HSV. IAC-HSV amplified in a non-stringent condition, ligated into pTZ57R plasmid vector, and transformed into Escherichia coli JM207 and then cloned. Resulting IAC was used for 105 CSF and 78 keratitis specimens.Results:PCR amplicons for HSV and IAC-HSV were 454-bp and 662-bp, respectively. Detection limit of IAC was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was 500 copies/mL of HSV DNA. Among all specimens, 7 inhibited specimens were detected.Conclusions:Indeed, using other DNA as an IAC is expected to detect false-negative results and amplification of the DNA is the key tool to examine the accuracy of amplification and detection steps. This internal amplification control is applicable for early reliable diagnosis of HSV in different loads of virus in different specimens.

Detection of peanut (Arachis hypogaea) allergen by Real-time PCR method with internal amplification control

Specific primer sets were designed based on the DNA sequence of Ara h 1, one of the major peanut (Arachis hypogaea) allergens, and a competitive internal amplification control (IAC) was designed by compound primer technology. By choosing 314copies/PCR as the IAC dosage, a Real-time PCR method with IAC was established for detecting peanut allergen Ara h 1 DNA. The method showed high specificity with a detection limit of 0.005% peanut. A series of commercial food products with/without peanut components were tested. Among these products, the peanut allergen Ara h 1 DNA could be detected in 12 products labelled containing peanut ingredients, in two without a declaration of peanut and one labelled that was produced in a facility that produced peanut-containing foods. This indicates that the method is highly sensitive for the detection of peanut ingredients in foods.

Asymmetric helicase-dependent amplification combined with lateral flow assay (HDA-LFA) for the detection of S. Paratyphi A

IntroductionS. Paratyphi A is a causative agent of paratyphoid fever with milder symptoms compared to typhoid fever. Culture method is the gold standard used for diagnosis and the disadvantages of the method is less sensitive, laborious and requires 3 – 5 days for identification of the bacteria. Therefore, a rapid and user-friendly asymmetric helicase-dependent amplification combined with lateral flow assay (HDA-LFA) was developed for the detection of S. Paratyphi A. ObjectivesTo determine the detection limit, sensitivity and specificity of the HDA-LFA for the detection of S. Paratyphi A using spiked stool samples. MethodsA pair of S. Paratyphi A HDA-LFA primer was designed to amplify a 93 bp intergenic region of S. Paratyphi A and a 123 bp competitive internal amplification control. The lateral flow strips were assembled after lining with antibodies using Biodot dispensing reagent. The asymmetric HDA assay was carried out using heating block for 1 hour at 65oC and the amplified products were then detected via LFA and analysed by naked eyes within 15 minutes.Results and discussion: The limit of detection for S. Paratyphi A HDA-LFA using spiked stool sample was at 102 CFU/ml while at 103 CFU/ml when using agarose gel electrophoresis method. The assay was also validated using stool samples spiked with 25 S. Paratyphi A and 75 non-S. Paratyphi A isolates and revealed 100% sensitivity and specificity without any inhibition. ConclusionThe assay is found to be rapid, sensitive and specific and has the potential to be an alternative DNA-based detection method for laboratories with limited access to specific equipment such as thermal cycler.

Development and evaluation of a multiplex PCR assay for simultaneous detection of major mycotoxigenic fungi from cereals

The aim of the present study was to develop a multiplex PCR (mPCR) assay for the simultaneous detection of five major metabolic pathway genes viz. aflr (Aflatoxin), pks (Ochratoxin A), tri5 (Trichothecene), pks13 (Zearalenone) and fum13 (Fumonisin), producing Aspergillus, Penicillium and Fusarium species. The mPCR assay with competitive internal amplification control to eliminate false negative results employing specific primers for each of the above mentioned five genes was optimized and validated using standard strains. The standardized mPCR assay detected all five major mycotoxin metabolic genes along with artificially inoculated maize seeds with mycotoxigenic Fusarium, Penicillium and Aspergillus spores. The detection limit of this mPCR assay was 1 × 103 spores per gram of artificially inoculated samples upon 48 h of incubation at room temperature. When the developed mPCR assay was applied on to 177 contaminated maize, paddy and sorghum, many of the samples (100 out of 177) were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high performance liquid chromatography and in general both the methods provided unequivocal results. The developed mPCR based assay was found to be rapid, cost effective and user friendly and can be used for diagnosis of major mycotoxigenic fungi.

Abstract 4150: Quantitative sequencing following PCR-driven library preparation with internal standard mixtures has improved analytical performance and lower cost.

Abstract Background: Next-generation sequencing (NGS) is amenable to a multitude of clinical applications by virtue of its automated and highly parallelized analysis of nucleic acid templates. However, prior studies have identified non-systematic biases introduced during preparation of NGS libraries as the primary source of technical variation preventing immediate application for measuring nucleic acid abundance in the clinical setting. We reasoned that a PCR-based NGS library preparation protocol that incorporated competitive internal amplification control (IAC) mixtures (i.e. internal standards) would control for the majority of bias introduced during NGS library preparation, enabling clinical laboratories to offer cost effective moderately complex diagnostic panels from quantitative NGS data. Methods: In order to test this approach, we obtained reference material RNA titration pools used in the FDA-sponsored Sequencing Quality Control (SEQC) project that have been characterized for nucleic acid abundance by multiple qPCR, Microarray and NGS platforms. Using Multiplex-PCR with primers and competitive IAC for 150-gene targets we prepared NGS libraries from: 1) gDNA to test general analytical performance, and 2) cDNA from reverse transcribed SEQC project reference material to determine accuracy in detecting fold change. Using gDNA mixed with serially titrated IAC mixtures as input, we observed a linear dynamic range over 106 orders of magnitude, with an average R2 = 0.995 (0.993 – 0.997; 95% CI). There was a high correlation coefficient (R2= 0.96) between measured values for copies of nucleic acid abundance in two separate library preparations (separate reverse transcriptions and Multiplex PCR-based library preparations) from the same reference RNA material (FDA SEQC project Sample A). Because the SEQC project RNA Samples C and D represent a known cross titration between SEQC project RNA Samples A and B, by comparing measured to expected values for expression of each gene in Samples C and D it is possible to determine accuracy of the method. In preliminary studies, the correlation coefficient of expected versus observed for Sample C was R2 = 0.96, with an ROC curve-determined accuracy to detect a 3-fold change of 97% (95 – 99%; 95% CI). Inter-platform and inter-laboratory comparisons are ongoing. Conclusion: The approach described here overcomes key sources of non-systematic bias introduced during NGS library preparation. This should enable reproducible inter-laboratory and inter-platform quantitative NGS results, and a clear path to regulatory approval for clinical diagnostic applications. Citation Format: Thomas Blomquist, Erin L. Crawford, James C. Willey. Quantitative sequencing following PCR-driven library preparation with internal standard mixtures has improved analytical performance and lower cost. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2013-4150

Taguchi optimization of duplex PCR for simultaneous identification ofStaphylococcus aureusandClostridium perfringensalpha toxins

Staphylococcus aureus and Clostridium perfringens are two major bacteria that infect open wounds and delay the healing process. The rapid and progressive deterioration of soft tissue during S.aureus and C.perfringens coinfections is due to analogous necrotic alpha toxins produced by the two organisms. The aim of this study was to determine the alpha toxins of S.aureus and C.perfringens by duplex PCR. The PCR assay employed two sets of primers: hlaf/r to amplify staphylococcal alpha toxin gene hla (274bp) and cpaf/r to amplify clostridial alpha toxin gene cpa (398bp) along with a competitive internal amplification control (608bp), simultaneously. Optimization of the duplex PCR assay was achieved by a modified Taguchi method, an engineering optimization process, in a nine-tube combinatorial array. The detection level of the duplex PCR was found to be 10pg of purified DNA or 10(3) CFUmL(-1) of S.aureus and 100pg of purified DNA or 10(4) CFUmL(-1) of C.perfringens. Other bacteria routinely found in tissue infections were tested for cross-reactivity and the duplex PCR turned out to be highly specific. This duplex PCR assay provides a rapid, robust and reliable alternative to the existing conventional techniques in establishing the aetiology of S.aureus and C.perfringens in soft tissue infections.

Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods

To develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of four major mycotoxin metabolic pathway genes, viz. nor1 (aflatoxin), Tri6 (trichothecene), FUM13 (fumonisin) and otanps (ochratoxin A). A mPCR assay with competitive internal amplification control, employing specific primers for each of the aforementioned four genes, was optimized and validated using 10 reference strains and 60 pure culture isolates. The standardized mPCR assay detected all four mycotoxin metabolic genes in artificially contaminated maize samples with a sensitivity of 2 × 10(3) CFU g(-1) for nor1-positive Aspergillus strains, Tri6 and FUM13-positive Fusarium strains and 2 × 10(4) CFU g(-1) for otanps-positive Penicillium strains. When the developed mPCR assay was applied to 40 natural foods, 35% (14 of 40) of the samples were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high-performance liquid chromatography (HPLC), and in general, both the methods provided unequivocal results. The current mPCR assay is a rapid and reliable tool for simultaneous specific and sensitive detection of aflatoxigenic Aspergillus strains, trichothecene- and fumonisin-producing Fusarium strains, and ochratoxigenic Penicillium species from naturally contaminated foods. This mPCR assay could be a supplementary strategy to current conventional mycotoxin analytical techniques such as thin-layer chromatography (TLC), high performance thin layer chromatography, HPLC, etc., and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.

Application of a Competitive Internal Amplification Control for the Detection of Sapoviruses in Wastewater

In this study, a competitive internal amplification control (IAC) was constructed for application in the real-time reverse transcription-polymerase chain reaction detection of sapoviruses (SaVs). A SaV RNA standard was also created for quantification of the virus. The IAC was included in the screening of environmental samples for SaVs. From August 2010 to December 2011, 51 wastewater samples were collected from five provinces in South Africa. SaVs were found in 72.5% (37/51) of samples, including four samples where detection was initially inhibited. SaV concentrations ranged from 4.24×10(3) to 1.31×10(6)copies/ml. The IAC successfully identified samples which contained inhibitors and inclusion of an IAC is necessary to ensure the prevalence of SaVs is accurately determined. SaVs are present at high concentrations in wastewater in several provinces of South Africa. This widespread occurrence indicates that SaV circulation in the South African population may be underestimated.

Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V. parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10(7) CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.