PCR Assay For Simultaneous Detection Research Articles

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

BackgroundThe blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies.MethodsPrimers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA.ResultsThe multiplex assay was 100% specific, with analytical sensitivities of 103 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA).ConclusionsThis multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.

A multiple reverse transcription PCR assay for simultaneous detection of four main viruses in kiwifruit

Kiwifruit (Actinidia spp.) is an important horticultural crop in the world. Previous studies showed that a number of viruses were found in kiwifruit around the world. To detect the multiple viruses in kiwifruit quickly and effectively, a multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed to detect four viruses simultaneously in kiwifruit: Actinidia chlorotic ringspot-associated virus (AcCRaV), Actinidia virus 1(AcV-1), Actinidia virus A (AcVA), Citrus leaf blotch virus (CLBV), respectively. Four pairs of primers were designed according to the coat protein (CP) genes of the aforementioned viruses and then were confirmed by RT-PCR assay. Actin gene (ACT1) was used as an internal control to improve reliability of the multiplex RT-PCR. Determination of detection limit of the multiplex RT-PCR showed that this method could detect four viruses at the dilution of 10−4 cDNA. Furthermore, the developed multiplex RT-PCR could detect viruses in kiwifruit field samples successfully.

Development and evaluation of a panel of multiplex one-tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions.

Multiplex real‐time quantitative polymerase chain reaction (mRT‐qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one‐tube nested real‐time PCR (mOTNRT‐PCR) assay consisting of five separate internally controlled RT‐qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT‐PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT‐PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real‐time PCR (RT‐qPCR) assay. The analytical sensitivities of mOTNRT‐PCR panel ranged from 2 to 20 copies/reaction, and no cross‐reaction with common respiratory viruses was observed. The coefficients of variation of intra‐assay and inter‐assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT‐PCR assay panel were missed by RT‐qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT‐PCR assay panel provides a more sensitive and high‐throughput method for the detection of 14 respiratory viruses.

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.

SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.

This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set. The RAPD primer, OPA4, amplified a specific fragment of c. 400bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5pgµl-1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas-specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas simultaneously in root tissues of citrus plants. A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots. A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.

Multiplex PCR assay for simultaneous detection and differentiation of Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in the municipality-supplied drinking water.

BACKGROUND:The contamination with Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in drinking water is the most prevalent in Indian subcontinent, but often difficult to detect all these pathogens from the drinking water.MATERIALS AND METHODS:A multiplex polymerase chain reaction (mPCR) method was developed to detect contamination of municipality-supplied drinking water with E. histolytica, G. lamblia, and Salmonella spp. The primers were designed to target small subunit of 16S rRNA type gene of E. histolytica and G. lamblia, and invasive A gene of Salmonella typhimurium. The optimized mPCR assay was applied on 158 municipality-supplied drinking water samples collected from Delhi.RESULTS:Out of total 158 water samples, 89 (56.32%) were found positive for the targeted pathogens by mPCR while conventional methods could be detected only in 11 (6.96%) samples. The mPCR assay showed 100% sensitivity and specificity for these pathogens in comparison with culture and microscopic detection. Of the 89 mPCR-positive samples, G. lamblia, E. histolytica, and Salmonella spp. were present in 35 (22.15%), 26 (16.45%), and 28 (17.72%), respectively. Nine (5.69%) samples were positive for both E. histolytica and G. lamblia, 10 (6.32%) were positive for G. lamblia and Salmonella spp., and 8 (5.06%) had Salmonella spp. and E. histolytica. Nonetheless, 3 (1.89%) samples were positive for all three pathogens.CONCLUSIONS:The present assay is an alternative to conventional methods to serve as highly sensitive, specific, and economical means for water quality surveillance to detect the outbreak caused by E. histolytica, G. lamblia, and Salmonella spp. pathogens.

A multiplex PCR assay for simultaneous detection of four major pathogenic bacteria of ayu

A multiplex PCR assay for simultaneous detection of four major pathogenic bacteria of ayu

Duplex PCR Assay for Identification of Tissue of Cattle and Buffalo Origin Targeting Mitochondrial Cytochrome b Gene

The aim of this study was to develop PCR assay for simultaneous detection of tissues from cattle and buffalo in a single PCR reaction. Species-specific primers for cattle and buffalo species were designed through homology comparisons and alignment of partial sequences of Cyt.b gene from common food animals. The conditions for species-specific PCR amplification were optimized in terms of quantity and concentration of various components for PCR mix and annealing temperature for each set of designed species-specific primer pairs. The assay revealed the amplification of genomic DNA from cattle to amplicon size of 655 bp, whereas DNA template from buffalo to amplicon length of 249 bp. Simultaneous amplification of DNA templates from cattle and buffalo was observed in duplex PCR assay. Thus, the developed assay could identify the tissues of cattle and buffalo origin, detecting even both the species together and being advantageous over species-specific PCR.

Development of multiplex PCR assay for simultaneous detection of five cucumber pathogens based on comparative genomics

The aim of this study was to develop a multiplex PCR assay for rapid, reliable and simultaneous detection of five important cucumber pathogens: Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Pythium aphanidermatum and Botrytis cinerea. One common forward primer and species-specific reverse primers were designed based on comparative genomics. The specificity of the developed multiplex PCR was confirmed using target pathogens and other closely related pathogens. Furthermore, this method accurately identified the presence of pathogens among cucumber field samples. Consequently, we established a multiplex PCR method which could provide reliable and informative identification of five cucumber pathogens in the tested environmental samples.

A multiplex reverse transcription PCR assay for simultaneous detection of six main RNA viruses in tomato plants

Tomato virus diseases occur all around the world, causing serious yield losses. To detect these viruses quickly and provide a basis for disease control, a multiplex reverse transcription polymerase chain reaction system was established for simultaneous detection of Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato chlorosis virus (ToCV), Potato virus Y (PVY) and Potato virus X (PVX) in tomato plants, with 6 pairs of specific primers being designed based on the coat protein (CP) genes of these viruses. Transcriptional elongation factor-1α (EF-1α) from tomato was added to the multiplex RT-PCR reaction system to prevent false negatives. The concentration of the primers, annealing temperature, annealing time, extension time and amplification cycles were optimized. Expected fragments of 159 bp (ToCV), 262 bp (PVY), 362 bp (EF-1α), 430 bp (TMV), 500 bp (TSWV), 600 bp (CMV) and 705 bp (PVX) were amplified by this multiplex RT-PCR system, and their origin was confirmed by DNA sequencing. This method will have a wide application in virus detection of field samples.

Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

Simultaneous detection of porcine pseudorabies virus, porcine parvovirus and porcine circovirus type 2 by multiplex real-time PCR and amplicon melting curve analysis using SYBR Green I

Porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 can cause reproductive failure in pigs, and swine are often simultaneously infected by combinations of the three viruses. We here report the development of a SYBR Green I-based multiplex real time PCR assay for simultaneous detection of porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2. Three pairs of specific primers were designed for the porcine parvovirus-VP2, porcine pseudorabies virus-gH and porcine circovirus type 2-ORF2 genes. Viral genomes were identified based on their distinctive melting temperatures in singleplex PCR reactions. The melting temperature was 74.5 °C for the 313 bp amplicon of porcine parvovirus-VP2 gene, 87.5 °C for the 355 bp amplicon of porcine pseudorabies virus-gH gene and 80.5 °C for the 171 bp amplicon of the porcine circovirus type 2-ORF2 gene, respectively. The detection limit of the method ranged from 0.01–0.03 TCID<sub>50</sub>/ml for the three viruses. In addition, porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 viral loads were measured in 100 field samples, and the result showed that the concordance between real-time PCR and conventional PCR was 60.42%. The sensitivity and specificity of real-time PCR were 100% and 100%, while those of conventional PCR were 40.83% and 72.22%, respectively.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides

BackgroundStreptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening.MethodsOne hundred two pregnant women who were at 35–37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses.ResultsThe real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7–99.6%), a specificity of 95.5% (86/90, CI 89.8–98.7%), a positive predictive value of 73.3% (11/15, CI 44.8–91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9–99.9%).ConclusionThe multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Development of a duplex PCR assay for simultaneous detection of Babesia bigemina and Theileria annulata infections in cattle

Bovine babesiosis and theileriosis are fatal tick borne haemoparasites of vertebrates imposing serious constraints on health and productivity of livestock. Additionally, the recovered animals become persistent carriers and play a significant role in disease epidemiology. The present investigation describes the development and evaluation of duplex PCR assay for simultaneous detection of Babesia bigemina (B. bigemina) and Theileria annulata (T. annulata) in cattle. Following in silico analysis for candidate target genes representing each of the haemoparasites, an optimised duplex PCR assay was established using two sets of primers, ssurRNA and cytob1 for genomic DNA amplification of B. bigemina and T. annulata encoding product size of 689 and 312 bp, respectively. The results were compared with conventional microscopy and monoplex PCR assay. The sensitivity of each primer pair was checked using serial dilutions of parasite DNA, while specificity was determined by testing for amplification from DNA of different stocks of each pathogen. The duplex PCR detected each parasite species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or with DNA mixture representing the other pathogens. Additionally, single and duplex PCRs could able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of both the pathogen, and nonspecific amplification from non target species was not observed. The developed assay represents an economical, simple, sensitive, specific and reproducible diagnostic tool for simultaneous detection of tropical theileriosis and bovine babesiosis and boosting targeted selective control strategy in endemic areas.

Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

A simple and sensitive two-tube multiplex PCR assay for simultaneous detection of ten meat species

A sensitive and reliable technique for meat species identification is required to prevent food adulteration, particularly in meat production. This work developed an optimized multiplex PCR assay for simultaneous identification of five commonly consumed and five commonly banned meat species in meat products. We designed primers that specifically amplified mitochondrial ATPase subunit 8 gene regions of different lengths of bovine, ovine, swine, chicken, turkey, cat, dog, mouse and human DNAs. The developed multiplex PCR assay proved to be specific, sensitive down to 30pg DNA per reaction, reproducible and economical. It could be used with a variety of raw meats and processed food samples and is easily applicable in a routine laboratory analysis without specific sophisticated equipment.

Development of a fast duplex real-time PCR assay for simultaneous detection of chicken and pigeon in raw and heat-treated meats

We developed a fast duplex real-time polymerase chain reaction assay based on TaqMan® probes for the simultaneous identification of chicken and pigeon meat in under 40 min. The primer sets used for both pigeon and chicken targeted the mitochondrial cytochrome b gene. To obtain simultaneous fluorescent signals, FAM and HEX probes were used to label the pigeon- and chicken-specific probes, respectively. This method specifically amplified chicken and pigeon DNA without any cross-reactivity to 20 animal species. The absolute detection limit was 0.1 pg of DNA extracted from both chicken and pigeon meat. Using DNA extracted from either raw or heat-treated meat, as little as 0.01% pigeon DNA was detectible in chicken DNA, as well as the reverse. This assay might serve as a rapid, sensitive, and specific detection method for the identification of pigeon and chicken meats in both raw- and cooked-meat products.

Heptaplex-direct PCR assay for simultaneous detection of foodborne pathogens

Foodborne pathogens pose significant problems for public health and economy. The gold standard, cultivation, is time-consuming and costly. In this study, a heptaplex-direct PCR assay for simultaneous detection of seven foodborne pathogens without DNA extraction and enrichment was developed and validated. Seven virulent genes of target strains were amplified and found that the assay provided the expected PCR fragment of 583, 490, 415, 343, 224, 209, and 105bp for Shigella spp., Shiga toxin-producing Escherichia coli (STEC), Streptococcus pyogenes, Campylobacter jejuni, Salmonella Typhi, Listeria monocytogenes, and Staphylococcus aureus, respectively. Validation study showed that the assay was highly reproducible, specific and sensitive (106–100CFU/ml of detection limit). Moreover, assay application on 22 artificially-contaminated and 100 food samples provided a statistically equivalent efficiency to the culture method. A heptaplex-direct PCR assay thus can be used in microbial forensic science.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P.damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P.damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

A novel real-time TaqMan™ PCR assay for simultaneous detection of Neotropical fox species using noninvasive samples based on cytochrome c oxidase subunit II

Strategies to evaluate and monitor elusive mammal species require the development of genetic techniques and their application to unambiguous biological material for ecological and genetic studies. In order to assess cytochrome c oxidase subunit II gene inter- and intraspecific variations, we compared sequences from different Neotropical canids and domestic dogs. We developed a primer pair to amplify a 154-bp fragment of this gene and a species-specific multiplex TaqMan™ assay for accurate identification of two native fox species occurring in sympatry in South America, the crab-eating fox (Cerdocyon thous) and the pampas fox (Lycalopex gymnocercus). The assays can also distinguish domestic dogs (Canis lupus familiaris) from both wild foxes. The use of different fluorescent reporter dyes for species identification in a multiplex probe PCR-RT assay reduces labor and costs. The methodology presented in this study demonstrates an efficient approach to enable high-performance analysis and represents a reliable cost-effective tool for molecular ecology research to monitor the wild canid populations by noninvasive genetic sampling. This standardized assay will allow large-scale high-throughput analyses in a routine and reliable way.