Internal Amplification Control Research Articles

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5ng–50fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 104CFU/g, this was improved to 10CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.

English

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis(MTB) which mostly affects the lungs. The disease causes deaths of many people every year. There are different methods to detect MTB such as skin test, staining, culture and molecular techniques. Polymerase chain reaction (PCR) is a simple and rapid method for the detection of MTB; however, positive and negative false results reduce the efficiency of this technique. The aim of this study was to design an internal amplification control (IAC) and apply it in MTB PCR test. PCR technique for MTB was optimized by using specific primers for IS6110 gene. The sensitivity and specificity of the test were determined. IAC was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on electrophoresis gel. The IC used in PCR testing of MTB is the competitive form in which the range was between 10 million and 10 bacteria and the most suitable internal control concentration for the mix was 1,000 plasmids. After making IC and using it in MTB amplification, it was observed that IC might guarantee the correctness of PCR reaction. Key words: Mycobacterium tuberculosis, polymerase chain reaction (PCR), internal control.

A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk

Escherichia coli and Shigella spp. are two major bacterial contaminants in raw milk. Effective screening of the two microbes before starting the production process is a critical step for the quality and safety guarantee of the resulting dairy products. This study reported a rapid and simple realtime PCR assay using a ubiquitous primer and probe set targeting the tuf gene for the detection of E. coli and Shigella spp. An internal amplification control (IAC) was also comprised to indicate false-negative results. The duplex realtime PCR assay showed a high efficiency above 96 % and a detection limit <10 cfu per PCR. When artificially contaminated raw milk samples were further evaluated, the assay performed equally as well as the traditionally cultural-based method, and facilitated quantitative detection of the two microbes in the range from ~102 to ~106 cfu mL−1 raw milk. The detection limit was ~102 cfu mL−1 raw milk for either or a mixture of the two strains without pre-enrichment step, and could be <10 cfu per 10 mL of raw milk if a pre-enrichment step was added. Considering the detection effectiveness, time-consumption saving, and economical efficiency, the present duplex realtime PCR assay has a great potential in the application in raw milk for assessing their microbiological quality and safety in relation to E. coli and Shigella spp.

Development of an immunomagnetic separation–propidium monoazide–polymerase chain reaction assay with internal amplification control for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk

A rapid, reproducible, sensitive and specific combination assay was developed for Escherichia coli O157:H7 comprising sequential immunomagnetic separation (IMS), followed by propidium monoazide (PMA), exclusion by viable cells, internal amplification control (IAC) and polymerase chain reaction (PCR). IMS was used to improve sensitivity, reduce detection time and eliminate false positives. PMA was used to detect viable cells, and IAC was incorporated into the system to eliminate the inhibitors of PCR from the food matrix. In the presence of inhibitors, IAC produced no signal, thereby eliminating false negative results. The results indicated that the IMS cell capture efficiency was about 90% at a concentration of 1 × 106 cfu mL−1 with a detection limit of 2.1 × 102 cfu mL−1 for pure culture. In an unoptimised system, IMS–PMA–PCR with IAC showed a detection limit of 5 × 103 cfu mL−1 in spiked milk over a 240 min assay, faster than conventional methods of detection.

Detection of allergenic parvalbumin of Atlantic and Pacific herrings in fish products by PCR

The conventional polymerase chain reaction (PCR) method to detect the major allergenic protein parvalbumin beta 2 of Atlantic herring (Clupea harengus) and Pacific herring (Clupea pallasii) was developed. The specific set of primers for the amplification of the partial genomic sequence of the pvalb 2 gene encoding the main fish allergen of both herrings was designed and applied to the investigation of 24 commercial fish products. The targeted amplicon size was 189 bp of pvalb 2 gene of Atlantic herring and Pacific herring. As the internal amplification control, the DNA of 18S rRNA gene for eukaryotes (141 bp) was successfully used. The specificity of designed primer pair using 26 various fish species was assessed. The intrinsic detection limit was 10 pg µl−1 of the present specific DNA. Atlantic herring or Pacific herring allergenic parvalbumins were detected in 22 investigated fish products in conformity with the package declaration. Two fish products were negative in spite of the declaration. The proposed PCR method is specific enough and can be used for the detection of Atlantic and Pacific herrings’ major allergen parvalbumin beta 2 in fish food products.

Specific and quantitative detection and identification of Cryptosporidium hominis andC. parvum in clinical and environmental samples

Cryptosporidium is an enteric protozoan parasite that is resistant to inactivation by commonly used drinking water disinfectants. Between 2004 and 2010, it was responsible for 60% of all waterborne protozoan parasitic outbreaks reported worldwide. Most sporadic infections in humans and almost all outbreaks are caused by Cryptosporidium parvum and Cryptosporidium hominis. We report the development and validation of a quantitative qPCR assay using minor groove binder (MGB)-probes targeting a unique Cryptosporidium specific protein-coding gene, that directly detects, quantitates and identifies C. hominis and C. parvum in environmental and faecal samples. An internal amplification control (IAC) was also developed and included in this assay. The qPCR assay was compared with an 18S nested PCR assay for sensitivity and specificity. The analytical sensitivity for the qPCR assay was 1 oocyst and 1–10 oocysts for the 18S assay. Evaluation of analytical specificity of the qPCR assay revealed no cross-reactions with other genera and detected all C. parvum and C. hominis isolates correctly. The diagnostic sensitivity and specificity of the qPCR was 100% compared to 96.9% and 98.4%, respectively for the 18S assay. The qPCR assay was also highly reproducible with RSD (relative standard deviation) values of 1.4–9.4%, when the assay was performed by four different technicians. When tested on water samples, the qPCR assay was more sensitive than the 18S assay, detecting positives in 37 of 138 water samples compared to 35 for the 18S locus. This qPCR assay should be a valuable tool for the detection and differentiation of C. hominis and C. parvum in both clinical and environmental samples.

Multiplex RT-PCR Reaction for Simultaneous Detection of Tomato Torrado Virus and Pepino Mosaic Virus Co-Infecting Solanum Lycopersicum

Abstract The tomato (Solanum lycopersicum L.) is cultivated all over the world and is a vegetable of significant economic importance. However, an increased production of the vegetable is directly connected with an elevated occurrence of pathogens limiting the production efficiency of the vegetable. Both, Tomato torrado virus and Pepino mosaic virus have been found to be serious disease factors. When not controlled, these viruses can significantly decrease tomato cultivation. In this article, we report a multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol for simultaneous detection of both, Tomato torrado virus (ToTV) and Pepino mosaic virus (PepMV) in virus infected plants. The assay was designed to specifically amplify the conserved regions of genomic ribonucleic acid (RNA) of both viruses. Moreover, the glycerandehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control of amplification to exclude false-negative assay results. High-resolution melt analysis of generated RT-PCR products was additionally performed to increase sensitivity and double-check the specificity of the reaction without the need of subsequent complementary deoxyribonucleic acid (cDNA) sequencing

Importance of Histamine-Producing Clostridium perfringens in Scombrotoxin-Forming Fish

It has been suggested that anaerobic histamine-producing bacteria (HPB) are important contributors to scombrotoxin fish poisoning (SFP). In order to assess the role of Clostridium perfringens in SFP, we developed a real-time PCR method for rapid detection of histamine-producing (HP) C. perfringens. The real-time PCR assay was 100% inclusive for detecting 23 HP C. perfringens and did not detect any of the other 116 HP or non-HP isolates examined. The efficiency of the assay with or without internal amplification control DNA was 102%; in the presence of background flora and inhibitory matrices, it was 90 to 99%. To investigate the importance of HP C. perfringens in SFP, we examined histamine production by C. perfringens in inoculated fish samples incubated under anaerobic conditions. C. perfringens produced low histamine levels in tuna (19 ppm) and Spanish mackerel (3 ppm), whereas gram-negative HPB produced high histamine levels (6,345 ppm in tuna; 1,223 ppm in Spanish mackerel) under the same conditions. When one bonito, two bigeye tuna, nine mahi-mahi, and five yellowfin tuna were examined for the presence of HPB, none (0 of 17) of the samples contained HP C. perfringens or other gram-positive HPB, whereas 86% of the samples contained gram-negative HPB. Our study indicates that histamine production by C. perfringens in scombrotoxin-forming fish was minimal compared with that by gram-negative HPB and that C. perfringens may not be an important bacterial species associated with SFP.

P2.048 Evaluation of Two Commercial Real-Time PCR Assays For Detection of Mycoplasma Genitalium in Urogenital Specimens

ObjectivesMycoplama genitalium is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. Nucleic acid amplification tests are currently the only available...

Evaluation of DAS™ kits for the detection of food-borne pathogens in chicken- and meat-based street-vended foods

The PCR-based method has gained acceptability as an alternative method for assessing the microbial safety of foods. In this study, the performance of PCR-based DNA Amplification System™ (DAS™) detection kits, namely, E. coli DAS™, S. aureus DAS™, E. coli O157:H7 DAS™ and Salmonella DAS™, were compared with reference methods in detecting the pathogens Escherichia coli, Staphylococcus aureus, E. coli O157:H7 and Salmonella spp. in 130 naturally- and artificially-contaminated chicken and meat-based grilled and fried street food samples. E. coli DAS™ displayed very good agreement with the reference method with a kappa value of 0.92 in chicken-based samples. S. aureus DAS™ also demonstrated very good agreement with the same kappa value of 0.92 but in meat-based samples. On the other hand, E. coli O157:H7 DAS™ and Salmonella DAS™ exhibited lower kappa values ranging from 0.67 to 0.76 in both food types. E. coli DAS™ obtained a high relative accuracy of 96% in chicken-based samples. S. aureus DAS™ also obtained a similar relative accuracy value of 96% but in meat-based samples. In terms of relative sensitivity, E. coli DAS™ and E. coli O157:H7 DAS™ showed high values of 96% and 95%, respectively, in chicken-based samples. Furthermore, E. coli DAS™, S. aureus DAS™ and Salmonella DAS™ demonstrated high relative specificities of 97%, 100% and 96% in chicken-, chicken-meat and meat-based samples, respectively. The performance of these kits was found to vary with food matrix, enrichment and cell-lysing procedures. Inclusion of internal amplification control, using more effective cell-lysing and enrichment procedures are suggested to enhance the overall performance of the kits.

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.

Safeguarding forensic DNA reference samples with nullomer barcodes

Unintended transfer of biological material containing DNA is a concern to all laboratories conducting PCR analysis. While forensic laboratories have protocols in place to reduce the possibility of contaminating casework samples, there is no way to detect when a reference sample is mislabeled as evidence, or contaminates a forensic sample. Thus there is public concern regarding the safeguarding of DNA submitted to crime labs. We demonstrate a method of introducing an internal amplification control to reference samples, in the form of a nullomer barcode which is based upon sequences absent or rare from publically accessible DNA databases. The detection of this barcode would indicate that the source of analyzed DNA was from a reference sample provided by an individual, and not from an evidence sample. We demonstrate that the nullomers can be added directly to collection devices (FTA paper) to allow tagging during the process of sample collection. We show that such nullomer oligonucleotides can be added to existing forensic typing and quantification kits, without affecting genotyping or quantification results. Finally, we show that even when diluted a million-fold and spilled on a knife, the nullomer tags can be clearly detected. These tags support the National Research Council of the National Academy recommendation that “Quality control procedures should be designed to identify mistakes, fraud, and bias” in forensic science (National Academy of Sciences, 2009).

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

Optimization and evaluation of a multiplex PCR for simultaneous detection of prominent foodborne pathogens of Enterobacteriaceae

Members of the family Enterobacteriaceae are major pathogens associated with gastrointestinal disorders caused by the consumption of contaminated foods. We have developed a multiplex PCR (mPCR) targeting specific genes for simultaneous detection and differentiation of five major Enterobacteriaceae members, namely, Salmonella sp. (invA), Escherichia coli (uidA), Shigella sp. (ipaH), Klebsiella pneumoniae (khe) and Citrobacter freundii (tpl), from both pure cultures and contaminated food samples, along with an internal amplification control (IAC). Simultaneous amplification of these five genes was optimized using reference strains and further evaluated on large number of isolates recovered from clinical and environmental sources. The mPCR assay showed high sensitivity for detecting 10 CFU/PCR for the above-mentioned pathogens directly from serially diluted overnight cultures. The mPCR assay was also able to detect all five pathogens spiked at an initial count of 10 CFU/g of meat and rice samples following an enrichment of 10 h in Brain Heart Infusion broth. To assess the practical application of this mPCR assay, we evaluated its efficacy for detecting possible contamination on natural samples, such as meat, fish, pastries and water. Based on the results, we suggest that this mPCR assay would be of immense help in detecting low counts of important Enterobacteriaceae pathogens inexpensively and thus can be used for the regular monitoring of food quality.

A Real-Time PCR/SYBR Green I Method for the Rapid Quantification of Salmonella enterica in Poultry Meat

It has been developed a method for quantitative detection of Salmonella enterica in poultry meat based on real-time PCR (qtPCR) with species-specific primers and SYBR® GreenER™ chemistry. Two methods for bacterial DNA extraction were compared: one based on a commercial kit (AccuPrep®) and the other on silica–magnetite nanoparticles. Primers were designed on sequence of invA gene encoding for an inner membrane protein associated with invasiveness of Salmonella. Serial dilutions of DNA from pure cultures of Salmonella and from broiler breast samples spiked with serial dilutions of Salmonella were analyzed in different replicates and with different PCR equipments. Robustness of the method was evaluated and compared in terms of repeatability, reproducibility, and consistency with conventional plate count methods and for applicability to the different equipments. The matrix effect upon each reaction specificity was assessed with addition of DNA from a noncompetitive internal amplification control. The limit of detection (LOD) was determined between 10 and 40 colony-forming units (CFUs)/ml; whereas, the limit of quantification (LOQ) was 102 CFUs/ml. Quantification with qtPCR was in the same order of magnitude as enumeration with plate counting but with an overestimation.

Inhibition of polymerase chain reaction for the detection of Escherichia coli O157:H7 and Salmonella enterica on walnut kernels

The aim of this study was to determine whether Escherichia coli O157:H7 can be reliably detected and isolated from walnut kernels using standard methods of analysis. The limit of detection approached 1 cell per analytical unit (25 g) for E. coli O157:H7 on walnut kernels enriched in modified tryptic soy broth with 20 μg/ml novobiocin and plating onto selective agar media. The presence of PCR inhibitors in walnut kernels was indicated by the failure to detect E. coli O157:H7 from culture positive enrichment broths analysed by PCR, with two separate polymerase and reagent compositions (Dupont BAX E. coli O157:H7 MP system, Promega GoTaq Green for stx) and three methods of template preparation (DuPont BAX, Qiagen DNeasy, Bio-Rad InstaGene). PCR inhibition was overcome by 1:100 dilution in TE buffer of the DNeasy or InstaGene template. PCR inhibition was not relieved by dilution of the BAX template. Similar results were observed for walnut kernels inoculated with Salmonella enterica and analysed for invA, indicating that PCR inhibition is not specific to the organism or primer/template. These results indicate that analysis of walnut kernels for pathogens should be with culture based methods or use protocols for DNA template preparation modified to remove or dilute inhibitors and the need for internal amplification controls in PCR methods.

Detection of Diarrheagenic Escherichia coli Using a Two‐System Multiplex‐PCR Protocol

Diarrheagenic Escherichia coli (DEC) strains are important causes of diarrhea. However, they cannot be distinguished from E. coli of the intestinal microbiota by conventional microbiological tests. This work presents a two-system multiplex PCR for detection of DEC. Primers for 16S rRNA gene were added as internal amplification control to validate negative reactions. The multiplex-PCR system 1 contains primers for detection of Shiga toxin producing E. coli (STEC; stx1, stx2), enteropathogenic E. coli (EPEC; eae, bfpA), atypical enteropathogenic E. coli (aEPEc; eae), enteroinvasive E. coli (ETEC; lt, st), enteroinvasive E. coli (EIEC; ial), and the internal amplification control 16S rRNA. The system 2 contains primers for EIEC (ipaH), enteroaggregative E. coli (CVD432), diffusely adherent E. coli (daaE), and 16S rRNA. The protocol was tested with E. coli reference strains, and also with cultures of fecal specimens of people with diarrhea and healthy controls. The protocol correctly identified the DEC reference strains. No DEC marker was amplified for negative controls; these results were validated by the amplification of a fragment of the 16S rRNA gene. The frequency of DEC was 7.6% for both patients and healthy controls; two Shigella sonnei strains were detected in the group with diarrhea. The identity of the amplicons was confirmed by DNA sequencing. The protocol is specific for DEC Shigella and is suitable for clinical laboratories.

Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei

BackgroundBurkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance.MethodsSignature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated.ResultsA single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction.ConclusionsThe new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

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.

Homogeneous duplex polymerase chain reaction assay using switchable lanthanide fluorescence probes

We have developed a duplex polymerase chain reaction (PCR) assay based on switchable lanthanide chelate complementation probes. In the complementation probe technology, two nonfluorescent oligonucleotide probes, one labeled with a lanthanide ion carrier chelate and another with a light absorbing antenna ligand, form a fluorescent complex by self-assembly of the reporter molecules when the two probes are hybridized in adjacent positions to the target DNA. Here we report the synthesis of a new terbium(III) (TbIII) ion carrier chelate and a new light-absorbing antenna ligand for TbIII and the development of a duplex Chlamydia trachomatis (Ct) PCR assay. For the detection of Ct in urine samples, a specific sequence in Ct cryptic plasmid was amplified and detected using europium(III) (EuIII) complementation probes. An internal amplification control was amplified in each reaction and detected using TbIII complementation probes to verify the Ct negative results. Ct bacteria were concentrated from urine samples with a rapid and simple centrifugation-based sample preparation method. Good diagnostic accuracy (99–100%) was achieved, and also Ct positive reactions yielded a very high EuIII signal-to-background ratio (maximum of 244). High performance of the complementation probes is advantageous when sample may contain impurities after a simple sample preparation.