Internal Amplification Control Research Articles

The presence and genetic characteristics of porcine circovirus 3 from pigs in Southern and Central provinces of Vietnam

Porcine circovirus type 3 (PCV3) is an emerging circovirus species that has recently been reported in different countries around the world, suggesting a widespread circulation. This study was carried out in order to investigate the presence and further genetic characteristics of PCV3 from swine herds in Southern and Central provinces of Vietnam. A duplex PCR assay for rapid detection of PCV3 in pigs was established with a pair of specific primers designed between rep and cap gene segment to amplify full-length ORF2 and another set of primers binding to COX1gene serving as an internal amplification control (IAC). The resulting duplex PCR was used to examine PCV3 presence in 94tissue and serum samples. Subsequently, PCV3 was detected in 10 out of 94 cases (10.6%). The infection rate in sows (14.3%) was higher than that in grower pigs (7.7%). Regarding nucleotide sequence comparison, 10 ORF2 genes were selected for nucleotide sequencing and their alignment showed 97.2% - 99.5% homology. According to the phylogenetic analysis and sequence alignment of cap gene, all the sequences were clustered into group PCV3a,including 9 strains of sub-group PCV3a1 and only one strain of subgroup PCV3a2. These findings indicated that the PCV3a group is circulating in swine farms in Vietnam. This study provides better insights into epidemiology of this pathogen in the national swine industry.

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne's Disease.

Johne's disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro

Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration-validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method.

Rapid Detection of Diarrheagenic Escherichia coli by a New Multiplex Real-Time Quantitative PCR Assay

Diarrheagenic Escherichia coli (DEC) is a group of important foodborne pathogens that can spread and cause infection in humans. As a significant public health issue, DEC infection may lead to a series of diseases such as abdominal pain, diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Therefore, its rapid and accurate detection has become an important concern for food safety issues and healthcare-associated infections. We have developed a multiplex real-time quantitative PCR assay (qPCR), DEC Sensor Test, to simultaneously recognize 5 major DEC pathotypes in a single tube, enterotoxigenic, enterohemorrhagic, enteropathogenic, enteroaggregative and enteroinvasive E. coli. In this assay, 13 DEC virulence genes, stp, sth, lt, stx1, stx2, eae, escV, bfpB, aggR, astA, pic, invE, and ipaH, can be simultaneously detected along with a spiked-in internal amplification control to avoid false negative results. All the constituent PCR for the 13 DEC virulence genes had high analytical sensitivity and specificity. Detection of all virulence genes by DEC Sensor Test can be much easier, cheaper and faster to perform than conventional tests. It also has added advantages of being able to distinguish live or dead bacteria as well as eliminating the possibility of switching partial results between different samples associated with multi-tube tests. Therefore, the application of this assay may significantly enhance our ability to prevent and fight DEC-associated diseases and to improve public health.

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula.

Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.

Two-Fluorophore Mobile Phone Imaging of Biplexed Real-Time NAATs Overcomes Optical Artifacts in Highly Scattering Porous Media.

Nucleic acid amplification tests (NAATs) are common in laboratory and clinical settings because of their low time to result and exquisite sensitivity and specificity. Laboratory NAATs include onboard positive controls to reduce false negatives and specialized hardware to enable real-time fluorescence detection. Recent efforts to translate NAATs into at-home tests sacrifice one or more of the benefits of laboratory NAATs, such as sensitivity, internal amplification controls (IACs), or time to result. In this manuscript, we describe a mobile-phone-based strategy for real-time imaging of biplexed NAATs in paper. The strategy consisted of: (1) using mobile phones with multipass excitation and emission filters on the flash and camera to image the signal from distinct fluorophore-labeled probe types in a biplexed NAAT in a glass fiber membrane; and (2) analyzing the differential fluorescence signal between the red and green color channels of phone images to overcome a strong evaporation-induced optical artifact in heated glass fiber pads due to changes in the refractive index. We demonstrated that differential fluorescence imaging enabled low limits of detection (316 copies of methicillin-resistant Staphylococcus aureus DNA) in our lab's "MD NAAT" platform, even in biplexed isothermal strand displacement amplification reactions containing 100k copies of coamplifying IAC DNA templates. These results suggest that two-fluorophore mobile phone imaging may enable translating the benefits of extant laboratory-based, real-time NAATs to the point of care.

Detection of toxigenic Vibrio cholerae, V. parahaemolyticus and V. vulnificus in oyster by multiplex-PCR with internal amplification control

This study developed a multiplex-polymerase chain reaction (m-PCR) method that targets the toxin genes, vvhA and vvp for Vibrio vulnificus, tdh for V. parahaemolyticus, ctx for V. cholera, to detect these three species simultaneously. A chimeric DNA consisting of a fragment of the green fluorescent protein gene (gfp) flanked by sequences of the vvhA primers was used as the internal amplification control (IAC). In the presence of these three Vibrio species, amplicons of IAC (753 bp), vvhA (505 bp), vvp (383 bp), tdh (256 bp) and ctx (167 bp) could be simultaneously detected. The accuracy of the m-PCR approach was evaluated with a Kappa index of 0.96 using 28 strains of V. vulnificus, 18 V. parahaemolyticus, 14 V. cholerae and 40 other Vibrio species and non-Vibrio strains. As little as 101 CFU/mL of these Vibrio species in spiked oyster homogenate could be detected by this m-PCR method following enrichment at 37°C for 8 h. This method can be adopted for the rapid detection of the toxigenic strains of Vibrio species in seafood.

Simultaneous detection of Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7 in environmental water using PMA combined with mPCR.

A multiplex polymerase chain reaction (mPCR) with propidium monoazide (PMA) and internal amplification control (IAC) for the simultaneous detection of waterborne pathogens Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7, was developed. This PMA-IAC-mPCR assay used four new specific primers based on the genes for invA, ecfX, cesB, and fliC, respectively. A 16S rRNA primer was chosen for IAC to eliminate false negative results. The photosensitive dye, propidium monoazide (PMA) was used to exclude signals from dead bacteria that could lead to false positive results. In pure culture, the limits of detection (LOD) were 101 CFU/ml for P. aeruginosa, 102 CFU/ml for both Salmonella spp. and E. coli O157:H7, and 103 CFU/ml for B. cereus, respectively. In addition, with a 6-8 h enrichment of all four bacteria that were combined in a mixture that was spiked in water sample matrix, the LOD was 3 CFU/ml for Salmonella spp., 7 CFU/ml for E. coli O157:H7, 10 CFU/ml for B. cereus and 2 CFU/ml for P. aeruginosa. This PMA-IAC-mPCR assay holds potential for application in the multiplex assay of waterborne pathogens.

Comparative study of multiplex real-time recombinase polymerase amplification and ISO 11290-1 methods for the detection of Listeria monocytogenes in dairy products

Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.

Improving Quantitative Power in Digital PCR through Digital High-Resolution Melting.

Applying digital PCR (dPCR) technology to challenging clinical and industrial detection tasks has become more prevalent because of its capability for absolute quantification and rare target detection. However, practices learned from quantitative PCR (qPCR) that promote assay robustness and wide-ranging utility are not readily applied in dPCR. These include internal amplification controls to account for false-negative reactions and amplicon high-resolution melt (HRM) analysis to distinguish true positives from false positives. Incorporation of internal amplification controls in dPCR is challenging because of the limited fluorescence channels available on most machines, and the application of HRM analysis is hindered by the separation of heating and imaging functions on most dPCR systems. We use a custom digital HRM platform to assess the utility of HRM-based approaches for mitigation of false positives and false negatives in dPCR. We show that detection of an exogenous internal control using dHRM analysis reduces the inclusion of false-negative partitions, changing the calculated DNA concentration up to 52%. The integration of dHRM analysis enables classification of partitions that would otherwise be considered ambiguous "rain," which accounts for up to ∼3% and ∼10% of partitions in intercalating dye and hydrolysis probe dPCR, respectively. We focused on developing an internal control method that would be compatible with broad-based microbial detection in dPCR-dHRM. Our approach can be applied to a number of DNA detection methods including microbial profiling and may advance the utility of dPCR in clinical applications where accurate quantification is imperative.

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.

A TaqMan-MGB real-time RT-PCR assay with an internal amplification control for rapid detection of Muscovy duck reovirus

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on TaqMan-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 101 copies/μL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay.The use of β-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established TaqMan-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity TaqMan-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection.

Multiplex reverse transcription-polymerase chain reaction for simultaneous detection of banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV) in abaca

The abaca (Musa textilis Nee) is an important fibre crop in the Philippines. Its fibre yield and quality is severely impacted by two potyviruses, namely, banana bract mosaic virus (BBrMV) and sugarcane mosaic virus (SCMV). A multiplex RT-PCR assay was developed for the simultaneous detection of these two RNA viruses in abaca. Primer sets were designed to amplify fragments of BBrMV coat protein (743 bp), SCMV coat protein (595 bp) and Musa sp. elongation factor 1 (219 bp) as an internal control. The specificity of each primer pair was confirmed by cloning and sequencing of the RT-PCR products. The multiplex RT-PCR assay was optimized by varying primer concentration combinations, dNTP concentration, Mg2+ concentration, Taq polymerase concentration, annealing temperature and extension temperature. The developed multiplex RT-PCR protocol has the same level of sensitivity and specificity when compared with uniplex RT-PCR detection for each virus and internal control. The inclusion of internal control amplification eliminates the risk of obtaining false negative results. The use of the optimized multiplex RT-PCR in simultaneous detection of BBrMV and SCMV from field samples proves that it is reliable, robust and faster than uniplex RT-PCR detection of each virus. This method will be useful for large-scale surveys of these two RNA viruses in abaca and will be vital for epidemiological studies and disease prevention.

RNA isolation efficacy of commercial and modified conventional methods for Citrus tristeza virus and mRNA internal control amplification

High quality RNA is critical for molecular and viral diagnostic applications. In case of citrus, polyphenol and polysaccharide rich content hinders many RNA isolation methods causing low quality RNA. These shortcomings can affect the Citrus tristeza virus (CTV) detection which is responsible to cause worldwide destruction of citrus orchards. Thus, in this study we analysed the RNA quality, quantity and integrity of different commercial and conventional methods. Commercial methods include TRIzol and kit-based method (PureLink RNA kit), while Cetyl trimethylammonium bromide (CTAB) and SDS/ Phenol (TENS-PCI) based method were optimized as conventional methods. For further validation, amplification potential was evaluated by performing Reverse transcriptase polymerase chain reaction (RT PCR) of CTV coat protein (p25) and mRNA internal control (nad5) genes to affirm RNA quality. Results indicated poor RNA recovery from TRIzol method with no successful amplification at RT PCR. Contrastingly, the modified conventional methods indicated RNA quality and purity for nucleic acids at Absorbance 260/280 (1.8–2.1) and Absorbance 260/230 (1.8–2.2) proving drastic improvement in RNA quality with reproducible amplification results. These results were further demonstrated with stored samples of different citrus varieties which indicated poor RNA integrity but successful RT PCR amplification. Consequently, our results may provide a brief comparison between isolation procedures by providing reference values to determine RNA purity requirements prior to RT PCR and help to determine rapidity, reproducibility and yield for each isolation method.

Detection of hepatitis E virus (rabbit genotype) in farmed rabbits entering the food chain

Hepatitis E virus (HEV) infects humans and many animal species. The rabbit HEV has been found in farmed, wild and pet rabbits as well as in human patients suggesting zoonotic transmission. Although the routes of human infection with rabbit strains are unclear a foodborne transmission is suggested especially when asymptomatically infected animals could enter the food chain. The aims of the study were an evaluation of the prevalence of HEV infections in slaughtered rabbits, identification of the virus genotype(s) and assessment of their genetic relatedness to other zoonotic HEV strains. A pair of blood and liver samples (n = 482) were collected from meat rabbits of different breeds slaughtered at the age of 2.8 to 6 months. The animals originated from 20 small-scale and 4 large-scale commercial farms operating in Poland. The presence of anti-HEV antibodies in animals was detected by the use of a recomWell HEV IgG (human) ELISA kit (Mikrogen Diagnostik) adapted to rabbit sera. The isolation of HEV and sample process control virus (feline calicivirus) RNA from homogenates of liver destined for food and virus-positive sera was performed using a QIAamp® Viral RNA Mini Kit (Qiagen). A one-step real-time reverse transcription PCR method containing a target-specific internal amplification control was used for detection of HEV. The (sub)genotype of detected rabbit HEV strains was identified based on sequence analysis of the ORF2 and ORF2/3 virus genome fragments. Anti-HEV antibodies were detected in 29 (6%) out of 482 rabbit sera samples collected from animals raised only on the small-scale rabbit farms. Four sera were also positive for HEV RNA. Viral RNA was detected in 72 (14.9%) animal livers. Analysing ELISA and PCR results using Student's t-test, there were significant differences observed in the frequency of HEV infections between rabbits from small-scale and commercial farms (t = 2.675, p = 0.015 < 0.05 for ELISA and t = 2.705, p = 0.014 < 0.05 for PCR). All detected virus strains were identified as HEV gt3 ra subtype. The results of this study provide data on the occurrence of HEV infections in rabbits entering the food chain, suggesting that a risk of foodborne HEV infection due to consumption of contaminated meat and liver exists. In this light, the presence of rabbit HEV in food animals is pertinent as an issue of food safety and the surveillance of these animals for emerging or re-emerging viruses.

Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

BackgroundEnterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings.MethodsDuplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays.ResultsThe limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively.ConclusionsThe developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

Development and evaluation of a duplex TaqMan qPCR assay for detection and quantification of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts

BackgroundA question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances.ResultsThe novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq’s) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq’s between 24 and 33.ConclusionsThis duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood

Bovine anaplasmosis is a tick-borne rickettsial disease, causing significant economic losses in many countries. The main causative agent of bovine anaplasmosis is Anaplasma marginale (Rickettsiales, Anaplasmataceae). To date, several PCR assays for A. marginale DNA detection were proposed, but most of them do not provide an internal amplification control, which allows to prevent false-negative results and is required for reliability of the results of pathogen DNA detection by PCR assay. In the present study, a real-time PCR assay based on the species-specific and highly conserved fragment of msp1α gene was developed for detection and quantification of A. marginale in bovine blood. The real-time PCR assay is able to detect as few as one copу of msp1α gene per reaction. To prevent false-negative results, simultaneous amplification and detection of the bovine genomic DNA fragment as an endogenous internal amplification control (IAC) was provided. The assay can be used as a highly specific and sensitive method for detection and quantification of A. marginale in infected cattle, and for the evaluation of the efficacy of anti-rickettsial drugs and anaplasmosis vaccines.