Exogenous Internal Positive Control Research Articles

Development and Clinical Applications of a 5-Plex Real-Time RT-PCR for Swine Enteric Coronaviruses.

A PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex real-time RT-PCR was developed and validated for the simultaneous detection and differentiation of four swine enteric coronaviruses (PEDV, PDCoV, TGEV and SADS-CoV) in one PCR reaction (XIPC serves as an exogenous internal positive control). The 5-plex PCR had excellent analytical specificity, analytical sensitivity, and repeatability based on the testing of various viral and bacterial pathogens, serial dilutions of virus isolates, and in vitro transcribed RNAs. The 5-plex PCR had comparable diagnostic performance to a commercial PEDV/TGEV/PDCoV reference PCR, based on the testing of 219 clinical samples. Subsequently, 1807 clinical samples collected from various U.S. states during 2019–2021 were tested by the 5-plex PCR to investigate the presence of SADS-CoV in U.S. swine and the frequency of detecting swine enteric CoVs. All 1807 samples tested negative for SADS-CoV. Among the samples positive for swine enteric CoVs, there was a low frequency of detecting TGEV, an intermediate frequency of detecting PDCoV, and a high frequency of detecting PEDV. Although there is no evidence of SADS-CoV presence in the U.S. at present, the availability of the 5-plex PCR will enable us to conduct ongoing surveillance to detect and differentiate these viruses in swine samples and other host species samples as some of these coronaviruses can cause cross-species infection.

Validation of a novel multiplex real-time PCR assay for Trypanosoma cruzi detection and quantification in açai pulp.

In Brazil, orally acquired T. cruzi infection has become the most relevant transmission mechanisms from public health perspective. Around 70% of new Chagas disease cases have been associated with consumption of contaminated food or beverages. Açai (Euterpe oleracea and Euterpe precatoria) is currently one of the most commercialized Amazonian fruits in the Brazilian and international markets. Therefore, it has become important to incorporate in the production process some procedures to measure out effective hygiene and product quality control required by global market. Molecular methods have been developed for rapid detection and quantification of T. cruzi DNA in several biological samples, including food matrices, for epidemiological investigation of Chagas disease and food quality control. However, a high-performance molecular methodology since DNA extraction until detection and quantification of T. cruzi DNA in açai berry pulp is still needed. Herein, a simple DNA extraction methodology was standardized from the supernatant of açai berry pulp stabilized in a 6M Guanidine-HCl/0.2M EDTA buffer. In addition, a multiplex real time qPCR assay, targeting T. cruzi DNA and an Exogenous Internal Positive Control was developed and validated, using reference from all T. cruzi DTUs and commercial samples of açai pulp, from an endemic municipality with previous history of oral Chagas disease outbreak. Thus, a high-sensitivity qPCR assay, that could detect up to 0.01 parasite equivalents/mL in açai, was reached. As of the 45 commercial samples analyzed, 9 (20%) were positive for T. cruzi. This high-sensitive, fast, and easy-to-use molecular assay is compatible with most of the laboratories involved in the investigations of oral Chagas disease outbreaks, representing an important tool to the epidemiology, control, and surveillance of Chagas disease.

Development of a universal internal positive control.

Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally designed, is small and efficiently amplified, has been successfully utilized alone or in triplex qPCR reactions, and is not crossreactive to human DNA or to any of the numerous non-human DNA samples tested.It was sensitive to inhibition by at least four commonly encountered amplification inhibitors. The IPC was used in a hydrolysis probe qPCR assay; however, it may be adaptable for other applications such as intercalating dye qPCR, PCR, isothermal amplification and reverse transcription-PCR.

Development and Application of a Multiplex qPCR Method for the Simultaneous Detection and Quantification of Pratylenchus alleni and P. penetrans in Quebec, Canada.

Root lesion nematodes are very common plant-parasitic nematodes that affect a wide range of plants. More than one species can be found simultaneously in a field, and each has a different impact on crop yield. Unfortunately, identifying them using classical morphometric criteria is very difficult and time consuming. The species Pratylenchus alleni was recently observed for the first time in Canada, associated with severe damage in a soybean field in the province of Quebec. The major species, P. penetrans, is also known to be endemic in Quebec but no data exist on its distribution in field crops. This prompted the development of a multiplex quantitative polymerase chain reaction (PCR) assay for the simultaneous detection and quantification of P. alleni and P. penetrans. The method was found to be specific and sensitive, systematically detecting a single larva in a 100-cm3 soil sample with no cross-amplification with other species, even when they outnumbered the target species. An exogenous internal positive control was included in the test to avoid false negatives due to the presence of PCR inhibitors. This assay was used to study the distribution of P. alleni and P. penetrans in 185 soybean fields in the major soybean-producing areas of Quebec during a 3-year survey. Overall, P. penetrans was found in 42% of the fields, P. alleni in 8%, and both species in 4%. The population density of P. alleni in positive fields was still very low, with only a few larvae detected. However, densities of P. penetrans were much higher: the provincial mean was 51.7 nematodes per 100 cm3 of soil (in positive samples), and 8% of the fields (15 of 185) exceeded the theoretical economic threshold. The presence of P. penetrans was also strongly correlated with soil texture, with lighter soil being the most favorable.

A simplified duplex real-time PCR incorporating TaqMan minor groove binder (MGB) probes and an exogenous internal positive control for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma synoviae cultures

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most pathogenic and economically important mycoplasma pathogens that infect chickens. The development of rapid and innovative molecular diagnostic techniques is of pivotal importance for their effective control. The aim of the present study was to develop a novel duplex TaqMan real-time PCR assay for the simultaneous detection of MG and MS. This duplex real-time PCR assay incorporates TaqMan (FAM/NED) labelled minor groove binder (MGB) probes that target the cytadhesin encoding surface protein (mgc2) gene and the haemagglutinin surface protein (vlhA) gene of MG and MS, respectively. The assay also contained a TaqMan exogenous internal positive control (Exo IPC), to avoid false negative results that might happen due to failure in DNA extraction/PCR inhibition. The TaqMan MGB probe-based duplex RT-PCR incorporating Exo IPC was then applied to DNA from culture isolates for the simultaneous detection of MG (mgc2 gene) and MS (vlhA gene). For duplex RT-PCR the sensitivity recorded was 10-3 CFU/ml and 10-2 CFU/ml for MG and MS template DNA, respectively. The specificity of the real-time PCR assay was 100% for MG- and MS-specific probes in detecting both single as well as double infections. In conclusion, the use of TaqMan MGB probes for the detection of mgc2 and vlhA genes confers extra specificity and the incorporation of Exo IPC simplifies the assay, allowing the detection of double infections with low-copy target DNAs.

Abstract 4978: Rapid and sensitive detection of oncoviruses using a simplified real-time RT-qPCR system

Abstract For cancers caused by oncoviruses, patients at risk can be identified before the disease has progressed to the stage of tumor formation. Traditional immunological detection methods have several drawbacks. In the case of Human Papillomavirus (HPV) which can cause cervical cancer, detection is difficult due to low expression of early viral proteins and a lack of sensitive and specific high-quality antibodies that can discriminate HPV types. Test results can also be misleading or inconclusive when a previous infection causes antibody levels to remain elevated in a patient from months to years afterwards, as in the case of the Epstein-Barr Virus (EBV), which is associated with Hodgkin's lymphoma and other carcinomas. Polymerase Chain Reaction (PCR) based detection of viral nucleic acids are becoming more prevalent in recent years due to better sensitivity, specificity, and ease-of-use than traditional methods. Sequence-specific probe binding adds one more layer of specificity to real-time PCR and has the additional advantage of providing viral load information. We have developed a simplified real-time Reverse Transcription, quantitative Polymerase Chain Reaction (RT-qPCR) system for the rapid detection of RNA and DNA samples that minimizes operator hands-on time to reduce error and risk of cross-contamination. HPV, EBV, Hepatitis B and C Virus (HBV and HCV) nucleic acid test controls were purified using conventional spin column methods, and the purified samples were amplified in a dilution series using the 4x TaqMan® Fast Virus 1-Step Master Mix (FVMM) (Life Technologies, CA) to evaluate sensitivity based on Cq, linearity, and efficiency. Common PCR inhibitors such as heparin, EDTA, hematin, and humic acid were spiked into reactions to evaluate the tolerance of the system based on ΔCq between inhibitor-spiked samples and control samples. High inhibitor tolerance would mean there is no need for discarding precious samples, re-purification, or dilution of samples with low abundance targets. Results show that 0.5 IU of HBV, 1.3 IU of HCV, and ∼10 copies of EBV can be detected per reaction. ΔCq of ≤1 is observed for Adenovirus and exogenous Internal Positive Control (IPC) assays when reactions are spiked with up to 0.0625 U heparin, 7.2 μg EDTA, 10 μM hematin, or 100 ng humic acid per reaction. This simple RT-qPCR system can be easily incorporated into any existing workflow. PCR is completed in less than 1 hour, making it ideal for screening tests. Multiplex feasibility as shown by the incorporation of an exogenous IPC in our experiments means users may combine both type identification and quantification of an oncovirus in a single reaction, further saving time, cost, and samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2011-4978

Development and application of a duplex QPCR for river water samples to monitor the myxozoan parasite Parvicapsula minibicornis

A duplex quantitative polymerase chain reaction (QPCR) assay was developed to simultaneously quantify the myxozoan parasite Parvicapsula minibicornis in river water samples and detect inhibition, which may compromise recognition of the target organism. The assay combines a TaqMan MGB probe specific to the nuclear small subunit ribosomal RNA gene of P. minibicornis and a commercial TaqMan Exogenous Internal Positive Control. P. minibicornis is endemic to freshwaters of the Pacific Northwest of North America and contributes to reduced fish health in Klamath River (Oregon/California) salmonids. The prevalence of P. minibicornis in these fish can reach 100%, and infection can result in glomerulonephritis and impaired kidney function. To better understand the temporal and spatial occurrence of this parasite in the Klamath River basin, water samples were taken from 7 mainstem sites and 5 tributaries along the 400 km river from March through September 2006. The samples were filtered, and the captured DNA was extracted and tested for the presence of P. minibicornis with the duplex QPCR assay. The parasite was present throughout the river over the entire sampling period, but its distribution and abundance varied spatially and temporally by over 2 orders of magnitude. Spore densities were lowest in March (spring) and peaked in June/July (summer) when site variance was also greatest. Inhibition levels also varied. The assay is able to detect 1 actinospore (the life cycle stage infective to fish) in 1 l of water and offers an alternative to sampling fish to monitor this pathogen and develop management options.

Specific and sensitive detection ofRalstonia solanacearumin soil with quantitative, real-time PCR assays

The aim of this study was to develop a sensitive and an effective method suitable for large-scale detection and quantification of Ralstonia solanacearum in soil. Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg microl(-1) DNA in conventional PCR and 1.2 fg microl(-1) in real-time PCR. By combining real-time PCR with the modified protocols to extract DNA from soil, it was possible to achieve real-time detection of R. solanacearum in soil, and the degree of sensitivity was 100 fg microl(-1). To detect inhibition in soil samples, an exogenous internal positive control (IPC) was included preventing false negative results, and IPC was successfully amplified from all samples tested. The methodology developed was used to detect the presence of R. solanacearum in tobacco fields in China. The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil. The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies.

Development and validation of a duplex real-time PCR assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples

A duplex real-time quantitative PCR (qPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and an exogenous internal positive control (IPC) in porcine semen samples was developed. The IPC was included to monitor DNA extraction and PCR inhibition and consisted of a mutated PCV2 plasmid clone which differed from the target PCV2 in the probe binding region and thus was detected by the use of a second probe with different end-labeling. The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested PCR (nPCR) methods for the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable tool for accurate and quantitative detection of PCV2 DNA in boar semen.

A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples

A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l −1 water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless ‘low contamination levels’ of water samples (<200 N. fowleri cells l −1) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.

Validation of Roche LightCycler Epstein-Barr Virus Quantification Reagents in a Clinical Laboratory Setting

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1–15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean™ Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond® spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log 10) calibration curve with a linearity range from 50 to 5 × 10 − 4 ng of DNA. A positive correlation ( R 2 = 0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.

Development of a novel internal positive control for Taqman® based assays

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.