Development Of Multiplex PCR Assay Research Articles

The development of multiplex PCR assays for the rapid identification of multiple Saccostrea species, and their practical applications in restoration and aquaculture

BackgroundThe ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted in the misidentification of species, creating challenges for understanding basic species-specific biological information that is required for restoration and aquaculture. Genetic barcoding has proven essential for accurate species identification and understanding species geographic ranges. To reduce the costs of molecular species identification we developed multiplex assays using the cytochrome c oxidase subunit I (COI or cox1) barcoding gene for the rapid identification of five species of oysters within the genus Saccostrea that are commonly found in Queensland, Australia: Saccostrea glomerata, Saccostrea lineage B, Saccostrea lineage F, Saccostrea lineage G, and Saccostrea spathulata (lineage J).ResultsMultiplex assays were successful in species-specific amplification of targeted species. The practical application of these primers was tested on wild spat collected from a pilot restoration project in Moreton Bay, Queensland, with identified species (S. glomerata, lineage B and lineage G) validated by Sanger sequencing. DNA sampling by extraction of oyster pallial fluid was also tested on adult oysters collected from the Noosa estuary in Queensland to assess whether oysters were able to be identified non-destructively. DNA concentrations as low as 1 ng/ μL still amplified in most cases, allowing for identification, and mortality at 6 weeks post pallial fluid collection was low (3 out of 104 sampled oysters).ConclusionThese multiplex assays will be essential tools for species identification in future studies, and we successfully demonstrate their practical application in both restoration and aquaculture contexts in Queensland. The multiplex assays developed in this study outline easily replicable methods for the development of additional species-specific primer sets for the rapid identification of other species of Saccostrea found across the Indo-Pacific, which will be instrumental in unravelling the taxonomic ambiguities within this genus in tropical regions.

Smart-Plexer: a breakthrough workflow for hybrid development of multiplex PCR assays

Developing multiplex PCR assays requires extensive experimental testing, the number of which exponentially increases by the number of multiplexed targets. Dedicated efforts must be devoted to the design of optimal multiplex assays ensuring specific and sensitive identification of multiple analytes in a single well reaction. Inspired by data-driven approaches, we reinvent the process of developing and designing multiplex assays using a hybrid, simple workflow, named Smart-Plexer, which couples empirical testing of singleplex assays and computer simulation to develop optimised multiplex combinations. The Smart-Plexer analyses kinetic inter-target distances between amplification curves to generate optimal multiplex PCR primer sets for accurate multi-pathogen identification. In this study, the Smart-Plexer method is applied and evaluated for seven respiratory infection target detection using an optimised multiplexed PCR assay. Single-channel multiplex assays, together with the recently published data-driven methodology, Amplification Curve Analysis (ACA), were demonstrated to be capable of classifying the presence of desired targets in a single test for seven common respiratory infection pathogens.

Development of multiplex PCR assay for species-specific detection and identification of Saprolegnia parasitica

Saprolegnia parasitica is the most important pathogen under the genus, Saprolegnia which causes devastating oomycete diseases in freshwater fish. At present, the most common molecular method for identification of Saprolegnia species is sequencing of ribosomal DNA internal transcribed spacer (rDNA-ITS) region. In this study, a highly sensitive multiplex PCR targeting rDNA-ITS region and a hypothetical protein gene was developed using two sets of primer pair. In this PCR, two amplicons of different size of 750 bp and 365 bp are produced only in case of S. parasitica while other Saprolegnia species had single amplicon. This protocol could also differentiate Saprolegnia species from other fungus based on the size of rDNA-ITS region. The protocol does not require sequencing and can identify S. parasitica in a single reaction. Therefore, the multiplex PCR developed in this study may prove to be an easier, faster and cheaper molecular method for identification of S. parasitica.

Development of multiplex PCR assay for simultaneous detection of African swine fever, porcine circo and porcine parvo viral infection from clinical samples

A diagnostic method for simultaneously detecting and distinguishing African Swine Fever (ASF), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV) in clinical specimens is critical for differential diagnosis, monitoring, and control in the field. Three primer pairs were designed and used to create a multiplex PCR assay. In addition, 356 porcine post mortem tissue samples from various parts of India's North Eastern region were tested by the developed multiplex PCR assay to demonstrate its accuracy. Using the designed primers, each of the ASF, PCV2 and PPV target genes was amplified, but no other porcine virus genes were detected. The assay's limit of detection was 102 copies/µl of PCV2, PPV, or ASFV. The detection of PCV2, PPV, and ASF in postmortem tissue samples revealed that they are co-circulating in India's North-Eastern region. The percentage positivity (PP) for PCV2, PPV and ASF single infection were 7.02% (25/356), 3.93% (14/356), and 3.37% (12/356), respectively, while the PP for PCV2& PPV co-infection was 2.80% (10/356), ASF & PCV2 co infection was 1.4% (5/356) and the ASF, PPV& PCV2 co-infection was1.40% (5/356). The results also indicate that the ASF can infect pigs alongside PCV and PPV.

Development of multiplex PCR assay for rapid detection Listeria monocytogenes in clinical samples

Listeria monocytogenes is widely present in the natural environment. This bacteria can cause infections in both humans and animals. In humans, the most vulnerable groups to be infected with L. monocytogenes are the elderly, people with an impaired immune system and chronically illness, pregnant women, and newborn babies. The aim of this study was to develop a multiplex PCR assay for the rapid detection of L. monocytogenes in mock clinical samples. A pair of primers were designed for detection of L. monocytogenes based on prs, a Listeria genus specific gene, and hly, a hemolysin gene. The specificity of the primers were tested by using different L. monocytogenes strains and other common pathogenic bacteria. The results showed that L. monocytogenes strains were positive in the detection and other tested strains were negative in mock (spiked) clinical samples. The sensitivity of multiplex PCR assay was 102 CFU/ml per reaction. The specificity and sensitivity of multiplex PCR technology for detecting L. monocytogenes in mock (spiked) clinical samples were high, and the assay could be completed within 1.5 hours. Therefore, this established multiplex PCR provides a rapid and reliable method and will be useful for the detection of L. monocytogenes in mock clinical samples.

Arboviruses in the Astrakhan region of Russia for 2018 season: The development of multiplex PCR assays and analysis of mosquitoes, ticks, and human blood sera.

The Astrakhan region of Russia is endemic for the number of arboviruses. In this paper, we describe the results of the detection of the list of neglected arboviruses in the Astrakhan region for the 2018 season. For the purpose of the study in-house PCR assays for detection of 18 arboviruses have been developed and validated using arboviruses obtained from Russian State Collection of Viruses. Pools of ticks (n=463) and mosquitoes (n=312) as well as 420 samples of human patients sera have been collected and analyzed. Using developed multiplex real-time PCR assays we were able to detect RNA of eight arboviruses (Crimean-Congo hemorrhagic fever virus, Dhori (Batken strain) virus, Batai virus, Tahyna virus, Uukuniemi virus, Inkoo virus, Sindbis virus and West Nile fever virus). All discovered viruses are capable of infecting humans causing fever and in some cases severe forms with hemorrhagic or neurologic symptoms. From PCR-positive samples, we were able to recover one isolate each of Dhori (Batken strain) virus and Crimean-Congo hemorrhagic fever virus which were further characterized by next-generation sequencing. The genomic sequences of identified Dhori (Batken strain) virus strain represent the most complete genome of Batken virus strain among previously reported.

Technical note: Development of multiplex PCR assays for the molecular characterization of Streptococcus uberis strains isolated from bovine mastitis

Streptococcus uberis is an important causative agent for clinical and subclinical mastitis in dairy cattle. The aim of this study was to develop 2 multiplex PCR assays (mPCR) for the simultaneous detection of virulence factors and housekeeping genes for use when investigating the genetic variability and distribution of Strep. uberis virulence factors. The tuf, cpn60, pauA, sodA, sua, oppF, and gapC genes were grouped in assay 1 (mPCR1) and the hasA, hasB, and hasC genes were included in assay 2 (mPCR2). The detection limits were 11.8 pg and 5.9 pg of DNA for mPCR1 and mPCR2, respectively. The 2 mPCR assays were validated with 56 Strep. uberis strains isolated from mastitis milk samples collected from different bovine herds in northern Italy. Results revealed that gapC and oppF were detected in 98.2% of the strains, whereas sua and hasC genes were detected in 94.6 and 89.2% of the strains, respectively. The most common pattern was gapC+, oppF+, cpn60+, sua+, sodA+, pauA+, tuf+, hasA+, hasB+, and hasC+, which appeared in 59% of the strains analyzed. The molecular assays developed in the present study represent a powerful tool for the evaluation of virulence pattern distribution in Strep. uberis strains associated with intramammary infections.

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.

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.

Development of Multiplex PCR Assay for Identification of Eight Species from Meats in Korea

Development of Multiplex PCR Assay for Identification of Eight Species from Meats in Korea

Development of multiplex PCR assay for authentication of Cornu Cervi Pantotrichum in traditional Chinese medicine based on cytochrome b and C oxidase subunit 1 genes

This study describes a method for discriminating the true Cervus antlers from its counterfeits using multiplex PCR. Bioinformatics were carried out to design the specific alleles primers for mitochondrial (mt) cytochrome b (Cyt b) and cytochrome C oxidase subunit 1 (Cox 1) genes. The mt DNA and genomic DNA were extracted from Cervi Cornu Pantotrichum through the modified alkaline and the salt-extracting method in addition to its counterfeits, respectively. Sufficient DNA templates were extracted from all samples used in two methods, and joint fragments of 354 bp and 543 bp that were specifically amplified from both of true Cervus antlers served as a standard control. The data revealed that the multiplex PCR-based assays using two primer sets can be used for forensic and quantitative identification of original Cervus deer products from counterfeit antlers in a single step.

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle.

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase ( dnaB) gene of R. anatipestifer, alkaline phosphatase ( phoA) gene of E. coli and invasion protein ( invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10 3 colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.

Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria

To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) region for timely patient management. Two mPCR assays were developed: Slow-Growers (SG) mPCR was used for the detection of slow-growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast-Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species-specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S-23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005-2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.

A Novel Real-Time Duplex PCR Assay for Detecting penA and ponA Genotypes in Neisseria gonorrhoeae: Comparison with Phenotypes Determined by the E-Test

For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved in the chromosomally mediated resistance to this antibiotic. To date, no development of multiplex PCR assays targeting a range of gonococcal genes and variations as a means of predicting antibiotic resistance has been reported. The aim of this study was to develop a duplex assay using DNA from isolated strains. We describe the development and evaluation on the LightCycler platform of a real-time duplex PCR assay (hybridization probe format) for rapid and specific detection of ponA and penA variations, predicting penicillin susceptibilities. The real-time duplex PCR assay successfully detected variations in ponA and penA genes by use of distinct melting temperatures from a total of 120 Neisseria gonorrhoeae isolates. Moreover, the variation profiles obtained with the real-time PCR and the melting analysis showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Nucleotide sequencing data were in complete agreement with multiplex assay results. The presented assay is suitable for the detection of chromosomally mediated resistant strains of Neisseria gonorrhoeae in genotyping studies and could be valuable in the effective antimicrobial strategy to gonococci.