Multiplex Polymerase Chain Reaction System Research Articles

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/μL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

Relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis.

To investigate relationship between vitreous interleukin-6 levels and vitreous particles findings on widefield optical coherence tomography in posterior uveitis. This retrospective study examined vitreous inflammatory cells (hyperreflective particles) of posterior uveitis on widefield optical coherence tomography (WOCT). We examined the number of hyperreflective particles (possibility of vitreous inflammatory cells) observed on WOCT and the correlations with interleukin-6 (IL-6) levels. The relationship between vitreous IL-6 levels and image findings from WOCT from 37 eyes (34 patients) with posterior uveitis were analyzed. Mean patient age was 63.4±15.7 years. (Mean± standard deviation) IL-6 concentration in vitreous humor was 79.9±7380.9 pg/mL Uveitis was infectious in 9 cases and non-infectious in 28 cases with multiplex polymerase chain reaction system. We measured the number and size of vitreous cells in the posterior vitreous, defined as the space between the upper vitreous and the internal limiting membrane on WOCT at the macular, upper, and lower regions. Image analysis software was also used for cell counting. A strong correlation was seen between human and software counts. Pearson's correlation coefficient (PCC) was performed to compare categorial variables (on macular +0.866; upper cavity +0.713; lower cavity +0.568; total vitreous cavity +0.834; P<0.001 each). IL-6 levels correlated with both vitreous cell counts and cell counts observed on macular WOCT (human-counted group +0.339, P = 0.04; software-counted group +0.349, P = 0.03). Infectious uveitis showed higher IL-6 levels (P = 0.016) and high cell counts compared with non-infectious uveitis (P = 0.04). Vitreous number of hyperreflective particles (cells) findings on WOCTcorrelated well with human and software cell counts. Vitreous cells findings on WOCT also correlated with IL-6 concentrations on macular.

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa.

The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as "effluxR detection assay" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.

Developmental validation of a novel multiple genotyping assay with 24 Canine STR loci

Canine individual identification and parentage testing are essential in various fields, including forensics and breeding programs. This study aimed to develop and validate the Canine 25 A kit, a multiplex polymerase chain reaction (PCR) system designed to address these critical requirements. This novel system enables the simultaneous amplification of 24 canine autosomal short tandem repeat (STR) loci and one sex-determining marker. Validation of the Canine 25 A kit was conducted following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, demonstrating significant sensitivity, high inhibitor tolerance, canine specificity within a mixture, species specificity, and precision in genotype determination. The Canine 25 A kit was crucial in resolving several forensic cases, such as casework samples from a dog attack incident and parentage determination. Its effectiveness in genotyping these samples highlights its significance in forensic applications. Population genetic parameter analysis revealed a high discriminatory power, as indicated by the calculated combined discrimination power (CDP) values for each breed exceeding 0.999 999 999 999, while the combined power of exclusion (CPE) surpassed 0.9999. Overall, the Canine 25 A kit offers a precise and dependable tool for canine individual identification and parentage determination.

Rapid identification of bacteria using a multiplex polymerase chain reaction system for acute abdominal infections.

Acute abdominal infections can be fatal if the causative organism (s) are misidentified. The spread of antimicrobial-resistant bacteria has become a serious problem worldwide, making antibiotic selection extremely difficult. Using quantitative metagenomic analysis, we evaluated a commercial multiplex polymerase chain reaction (PCR) system (FilmArray™, bioMérieux, Marcy-l'Étoile, France) for the rapid identification of causative bacteria. The cases of 10 patients with acute abdominal infections were enrolled in this retrospective study. There were six cases of perforated peritonitis and four cases of intraabdominal abscess. Fluid collected from the acute surgical abdominal infections were examined. All specimens tested positive for microorganisms in culture, and six involved two or more microorganisms. Using the multiplex PCR system, nine of ten specimens were found to involve at least one microorganism. One specimen was not included in the multiplex PCR system panel. Nineteen of 21 microorganisms (90.5%) isolated by culture were detected by the multiplex PCR system. Microorganisms with very small numbers of reads (19 reads) were detectable. This multiplex PCR system showed a high detection rate for causative microorganisms in ascites and intraabdominal abscesses. This system may be suitable as an affordable rapid identification system for causative bacteria in these cases.

Evaluation of Legionella pneumophila SGUT Serotypes Isolated from Bath Water Using a Multiplex-PCR Serotyping Assay.

Legionella pneumophila, the primary causative agent of Legionnaires' disease, is classified into at least 15 serogroups (SGs). Before genotyping, serotyping is first performed to limit the sources of L. pneumophila infections that caused an outbreak. In addition to conventional assays using monoclonal or polyclonal antisera, serotyping using multiplex polymerase chain reaction (M-PCR) was recently developed for L. pneumophila. In this study, we applied the M-PCR system to 41 strains that remained to be SGUT (untypable) by slide agglutination tests among the 220 L. pneumophila strains isolated from bath water in Kobe City during 2016-2020, to determine SG-genotypes (SGg) by PCR amplification of the specific target gene of the SGs. Among the 41 SGUT strains, SGg4/10/14 was the most predominant (24/41, 58.5%), followed by SGg1 (7/41, 17.1%). Seven strains, except for the strains determined as SGg1, were identified as belonging to a single SGg by M-PCR serotyping (SGg5 [3/41, 7.3%], SGg8 [3/41, 7.3%], and SGg7 [1/41, 2.4%]). Furthermore, we found that the seven strains identified as SGg1 harbored particular genotypes. In conclusion, the M-PCR serotyping assay will be helpful for investigating the distribution of L. pneumophila in environmental and clinical settings.

Forensic Validation Studies of a Novel 35-InDel Multiplex Polymerase Chain Reaction System

Abstract Background: A difficulty associated with forensic applications is the detection of degraded biological materials. Due to the large amplicon sizes of short tandem repeat alleles, valid genotyping results cannot be obtained from degraded biological materials. Recently, insertion/deletion (InDel) polymorphisms have been used in forensic applications for their widespread distributions in the human genome, short amplicon sizes, and low mutation rates. Purpose: Human identification InDel panels have mostly been designed for European populations. Therefore, our laboratory independently developed a multiplex polymerase chain reaction (PCR) system with 35 polymorphic InDel loci to be used for human identification in China. Forensic validation studies were conducted on this novel 35-InDel multiplex PCR system. Methods: The 35 InDel loci were screened in the database, and then used with the traditional PCR amplification and capillary electrophoresis platform combined with five-color fluorescence parallel detection technology. Validation studies were performed on this novel panel, including accuracy, repeatability and reproducibility, species specificity, sensitivity, stability, forensic case sample detection, and mixture studies. In addition, forensic efficiency assessments were conducted in populations from different continents. Results: The data of validated studies indicated that the novel 35-InDel panel was accurate, stable, and efficient for forensic purposes. For human identification, the cumulative power of discrimination values for the these 35 InDel loci in East Asian, South Asian, European, American, and African populations were 0.999999999999995, 0.999999999999995, 0.999999999999971, 0.9999999999999960, and 0.999999999998166, respectively. Conclusions: In this study, a set of 35 InDel loci were conducted in a multiplex amplification system for human identification of degraded DNA sample, and this new assay was efficient and stable. The present results suggested that the 35-InDel panel was a reliable tool for forensic use and could be efficiently used for human identification in the East Asian populations.

The Detection of Foodborne Pathogenic Bacteria in Seafood Using a Multiplex Polymerase Chain Reaction System

Multiplex polymerase chain reaction (PCR) assays are mainly used to simultaneously detect or identify multiple pathogenic microorganisms. To achieve high specificity for detecting foodborne pathogenic bacteria, specific primers need to be designed for the target strains. In this study, we designed and achieved a multiplex PCR system for detecting eight foodborne pathogenic bacteria using specific genes: toxS for Vibrio parahaemolyticus, virR for Listeria monocytogenes, recN for Cronobacter sakazakii, ipaH for Shigella flexneri, CarA for Pseudomonas putida, rfbE for Escherichia coli, vvhA for Vibrio vulnificus, and gyrB for Vibrio alginolyticus. The sensitivity of the single system in this study was found to be 20, 1.5, 15, 15, 13, 14, 17, and 1.8 pg for V. parahaemolyticus, L. monocytogenes, E. coli O157:H7, C. sakazakii, S. flexneri, P. putida, V. vulnificus, and V. alginolyticus, respectively. The minimum detection limit of the multiplex system reaches pg/μL detection level; in addition, the multiplex system exhibited good specificity and stability. Finally, the assays maintained good specificity and sensitivity of 104 CFU/mL for most of the samples and we used 176 samples of eight aquatic foods, which were artificially contaminated to simulate the detection of real samples. In conclusion, the multiplex PCR method is stable, specific, sensitive, and time-efficient. Moreover, the method is well suited for contamination detection in these eight aquatic foods and can rapidly detect pathogenic microorganisms.

Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China.

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152bp (TMV), 205bp (ZYMV), 318bp (WMV), 419bp (CGMMV), 529bp (CMV), 662bp (A. citrulli), and 821bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

Multiplex PCR assay based on the citE2 gene and intergenic sequence for the rapid detection of Salmonella Pullorum in chickens

Salmonella is one of the most common Gram-negative pathogens and seriously threatens chicken farms and food safety. This study aimed to establish a multiplex polymerase chain reaction (PCR) approach for the identification of different Salmonella enterica subsp. enterica. The citE2 gene and interval sequence of SPS4_00301–SPS4_00311 existed in all S. enterica subsp. enterica serovars by genomic comparison. By contrast, a 76 bp deletion in citE2 was found only in Salmonella Pullorum. Two pairs of special primers designed from citE2 and interval sequence were used to establish the multiplex PCR system. The optimized multiplex PCR system could distinguish Salmonella Pullorum and non-Salmonella Pullorum. The sensitivity of the optimized multiplex PCR system could be as low as 6.25 pg/μL and 104 colony-forming units (CFU)/mL for genomic DNA and Salmonella Pullorum cells, respectively. The developed multiplex PCR assay distinguished Salmonella Pullorum from 33 different Salmonella enterica subsp. enterica serotypes and 13 non-target species. The detection of egg samples artificially contaminated with Salmonella Pullorum, Salmonella Enteritidis, and naturally contaminated 69 anal swab samples showed that results were consistent with the culture method. These features indicated that the developed multiplex PCR system had high sensitivity and specificity and could be used for the accurate detection of Salmonella Pullorum in clinical samples.

Development of SSR and SNP markers for identifying opium poppy.

Papaver somniferum L. (opium poppy) is the original plant of heroin, which is a major narcotic drug, and this plant has brought great harm to human health. However, the ban on opium poppy cultivation and trafficking is facing great challenges because of abnormal profits. Therefore, rapid and accurate identification is important to address the abovementioned problems. In this study, eleven simple sequence repeats (SSR) markers and two single nucleotide polymorphism (SNP) markers were mined to distinguish opium poppy from other six Papaver species. These molecular markers were further verified through a large number of plant materials of these seven Papaver species. An excellent multiplex polymerase chain reaction (PCR) system that simultaneously amplifies the three of eleven SSR markers was developed, which effectively improves the efficiency and speed of identification. The present research is of great implication for identifying and investigating the illegal cultivation and trafficking of opium poppy.

Development of a molecular marker based on chloroplast gene for specific identification of Korean Hibiscus (Hibiscus syriacus \u2018Simbaek\u2019)

Due to the rise in substitution and adulteration of herbal products, as well as the lack of genetic information on Hibiscus plants, more molecular markers are needed to understand the genetic diversity and avoid their misidentification. There are many allelic variants of the functional genes in Hibiscus and other plants, which control their respective phenotypes and other characteristics. Identifying alleles of the desired trait by determining diversification through gene-typing allele-specific markers for authentication is, therefore, a potent strategy. The purpose of this study was to use insertion/deletion (InDel) markers to identify Hibiscus syriacus cultivars. We developed a novel InDel marker for Korean Hibiscus ‘Simbaek’, based on the trnL-F region of the chloroplast gene. Through this InDel site, a modified specific primer pair and a novel multiplex polymerase chain reaction (PCR) system were developed for specific identification of the Korean Hibiscus Simbaek cultivar. The molecular markers developed in this study were highly specific and accurately authenticated as Simbaek from the five main cultivars of H. syriacus. Taken together, the described method is a potential tool for the identification and selection of germplasm resource of Simbaek cultivar.Graphical

Development of Multiplex PCR Coupled DNA Chip Technology for Assessment of Endogenous and Exogenous Allergens in GM Soybean.

Allergenicity assessment of transgenic plants and foods is important for food safety, labeling regulations, and health protection. The aim of this study was to develop an effective multi-allergen diagnostic approach for transgenic soybean assessment. For this purpose, multiplex polymerase chain reaction (PCR) coupled with DNA chip technology was employed. The study was focused on the herbicide-resistant Roundup Ready soya (RRS) using a set of certified reference materials consisting of 0, 0.1%, 0.5%, and 10% RRS. Technically, the procedure included design of PCR primers and probes; genomic DNA extraction; development of uniplex and multiplex PCR systems; DNA analysis by agarose gel electrophoresis; microarray development, hybridization, and scanning. The use of the asymmetric multiplex PCR method is shown to be very efficient for DNA hybridization with biochip probes. We demonstrate that newly developed fourplex PCR methods coupled with DNA-biochips enable simultaneous identification of three major endogenous allergens, namely, Gly m Bd 28K, Gly m Bd 30K, and lectin, as well as exogenous 5-enolppyruvyl shikimate-phosphate synthase (epsps) expressed in herbicide-resistant roundup ready GMOs. The approach developed in this study can be used for accurate, cheap, and fast testing of food allergens.

Multiplex fluorescence quantitative PCR for the detection of eight bacterial pathogens of porcine respiratory disease complex (PRDC)

As rapid and reliable tools for diagnosis of porcine respiratory disease complex (PRDC) bacterial pathogens are getting more important over time, we established a new multiplex fluorescence quantitative polymerase chain reaction (mFQ-PCR) system with a similar symptom. Our results showed that the system could accurately and simultaneously detect 8 pathogens within 1.5 h. The detection limits were 2-5 standard DNA copies. The correlation coefficient of the standard curve was more than 0.998, with a well dynamic range from 101 to 109, and the intra- and intervariation coefficients (CVs) were less than 2.06%. After setting the positive threshold of Ct value, the coincidence rate between mFQ-PCR and conventional PCR (cPCR) could reach 92%-100%, the specificity and sensitivity of mFQ-PCR exceeded 93.5% and 90.8%, respectively. We also found that the positive rates of 8 bacteria were 12.5%-68.2%, while the rates of co-infection were 90%-100% in the clinical samples. The co-infection among Streptococcus suis (Ss), Haemophilus parasuis (Hps), and Mycoplasma hyorhinis (Mhr) accounted for 78.3%, 91.4%, 100%, respectively of all the positive samples, among Actinobacillus pleuropneumoniae (App) and Mycoplasma pneumoniae (Mhp) are 45.5% and 35.7%, among Bordetella bronchis (Bb), Pasteurella multocida (Pm) and toxic Pasteurella multocida (T$^{+}$Pm) are 52.2%, 41.4%, and 100%, respectively. The results demonstrated that co-infection of 2-4 pathogenic bacteria with similar symptoms was a common situation for PRDC in south of China; our method has the potential to become a reliable detection tool.

The African Network for Improved Diagnostics, Epidemiology and Management of common infectious Agents

BackgroundIn sub-Saharan Africa, acute respiratory infections (ARI), acute gastrointestinal infections (GI) and acute febrile disease of unknown cause (AFDUC) have a large disease burden, especially among children, while respective aetiologies often remain unresolved. The need for robust infectious disease surveillance to detect emerging pathogens along with common human pathogens has been highlighted by the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. The African Network for Improved Diagnostics, Epidemiology and Management of Common Infectious Agents (ANDEMIA) is a sentinel surveillance study on the aetiology and clinical characteristics of ARI, GI and AFDUC in sub-Saharan Africa.MethodsANDEMIA includes 12 urban and rural health care facilities in four African countries (Côte d’Ivoire, Burkina Faso, Democratic Republic of the Congo and Republic of South Africa). It was piloted in 2018 in Côte d’Ivoire and the initial phase will run from 2019 to 2021. Case definitions for ARI, GI and AFDUC were established, as well as syndrome-specific sampling algorithms including the collection of blood, naso- and oropharyngeal swabs and stool. Samples are tested using comprehensive diagnostic protocols, ranging from classic bacteriology and antimicrobial resistance screening to multiplex real-time polymerase chain reaction (PCR) systems and High Throughput Sequencing. In March 2020, PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and analysis of full genomic information was included in the study. Standardised questionnaires collect relevant clinical, demographic, socio-economic and behavioural data for epidemiologic analyses. Controls are enrolled over a 12-month period for a nested case-control study. Data will be assessed descriptively and aetiologies will be evaluated using a latent class analysis among cases. Among cases and controls, an integrated analytic approach using logistic regression and Bayesian estimation will be employed to improve the assessment of aetiology and associated risk factors.DiscussionANDEMIA aims to expand our understanding of ARI, GI and AFDUC aetiologies in sub-Saharan Africa using a comprehensive laboratory diagnostics strategy. It will foster early detection of emerging threats and continued monitoring of important common pathogens. The network collaboration will be strengthened and site diagnostic capacities will be reinforced to improve quality management and patient care.

Study on bacterial pathogens through multiplex polymerase chain reaction system and their antimicrobial resistance pattern in goats presumed with fever and/or diarrhea.

Background and Aim:Goat is one of the major livestock species, plays an important role in the economy of Bangladesh. However, the outbreak of different infectious diseases in goats causes high mortality and economic losses due to lack of proper diagnosis and treatment. Conventional culture-based methods for detecting specific pathogens as confirmatory diagnosis are laborious as well as time-consuming in comparison to multiplex polymerase chain reaction (mPCR), by which multiple pathogens can be detected at a time. The present study was aimed to perform faster molecular identification of bacterial pathogens from goats presumed with fever and/or diarrhea and their antimicrobial resistance (AMR) pattern.Materials and Methods:A total of 200 blood samples were collected from goats at S. A. Quaderi Teaching Veterinary Hospital (SAQTVH) in Chattogram Veterinary and Animal Sciences University for the period of July 2017-April 2018. DNA was extracted and subsequently, mPCR assay was performed for the screening of several bacterial pathogens (Salmonella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli, Clostridium perfringens, Vibrio cholerae, and Staphylococcus aureus). An antimicrobial susceptibility test against ten antimicrobials for positive samples of each organism was conducted using the Kirby–Bauer Disk-Diffusion Method on selective media.Results:S. aureus, C. perfringens, L. monocytogenes, and Salmonella spp. were detected from collected samples and their overall prevalence was 11.5%, 3.5%, 1%, and 20.5%, respectively. The most common clinical signs were mild fever, nasal discharge, dyspnea, and coughing (39.1%) for S. aureus, diarrhea, convulsion, abdominal pain, and incoordination (57.1%) for C. perfringens, fever, protrusion of tongue, and incoordination (100%) for L. monocytogenes, and fever, anorexia, dehydration with mucous feces (36.6%) for Salmonella spp. infection in goats. AntimGentamicinicrobial diagram of S. aureus showed resistance against Cefotaxime (74%), Cefixime (65%), and Tetracycline (65%); highly sensitive against Amoxicillin (48%), Ciprofloxacin (44%), and Gentamicin (44%). On the other hand, C. perfringens showed highly resistant against Ampicillin (71%), Gentamicin (71%), sensitive against Penicillin (57%), and Cefotaxime (57%). L. monocytogenes were found to be sensitive to Penicillin (100%) and Cefixime (100%) and Salmonella spp. showed resistance to Ampicillin (78%) and Amoxicillin (59%) but sensitive to Ciprofloxacin (54%).Conclusion:This study identified pathogens with their specific clinical signs in goats presumed fever and/or diarrhea through mPCR with their AMR pattern in SAQTVH, Chattogram. Potential risk factors, measuring the strength of association of disease caused by these particular pathogens, were also determined. mPCR may use as an effective tool for rapid detection of pathogens in animal.

P135 Undifferentiated round cell and spindle cell sarcomas: the current paradigm

IntroductionThe classification of soft tissue and bone neoplasms is experiencing a transformation as methods to detect gene fusions expand. Next-generation sequencing panels, anchored multiplex polymerase chain reaction systems and comprehensive RNA sequencing have all contributed to new histological categorisation. The EWSR1-NFATC2 is one such novel translocation found in a round cell sarcomas. These sarcoma sit within the WHO group of undifferentiated round and spindle cell sarcomas. They presents as either a primary bone or soft tissue tumour and exhibits distinctive histopathologic features.MethodsThis is a case study of a soft tissue mesenchymal tumour with the recently described gene fusion EWSR1-NFATC2. The case is described, with 3-month outcomes and considered in the most recent literature on this sarcoma subset (26 cases).ResultsThis case study prompted a look at the current NHS paradigm for these emerging histopathologically distinctive sarcoma subgroups. At present the expertise with the sarcoma MDT, along with second opinion from other national MDTs is the format for subtype specific sarcoma management, informed by recent literature.ConclusionThe EWSR1-NFATC2 fusion round cell sarcoma tumours are an example of a very recently emerged sarcoma subset which display oncological variance to the wider Ewing’s family. The novel literature guided the Southmead and Royal Marsden MDT opinions to proceed straight to surgical excision.The Authors recommend a national prospective database for rare sarcoma subsets, particularly those that appear to exhibit distinctive clinicopathology characteristics. The format could be through the RSTN collaborative, secure anonymised data collection can be collected through REDCap.

Simultaneous detection of four lily-infecting viruses by a multiplex RT-PCR assay

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) system was modified and evaluated for the simultaneous detection of multiple viruses in coinfected lily plants. Four major lily viruses, namely, lily symptomless, cucumber mosaic, lily mottle, and plantago asiatica mosaic viruses, were targeted by simultaneous detection. Analysis of PCR products confirmed the amplification of virus-specific DNA fragments of the expected sizes and nucleotide sequences from the target viruses. The cDNAs from singly infected plant samples were used as positive controls, and multiple infected samples were used to develop the multiplex RT-PCR system, which was then applied to leaf samples from diverse lily species and hybrids. The modified multiplex RT-PCR was successfully validated on the simultaneous detection of these viruses.

Development of a multiplex microsatellite assay for parentage assignment in orange‐spotted groupers (Epinephelus coioides)

AbstractEstablishment of reliable pedigree information of different lineages is crucial for selective breeding and stock enhancement programs. However, owing to the absence of a reliable assay for parentage assignment, such information is lacking in orange‐spotted grouper (Epinephelus coioides), which is an economically important coral‐reef fish. In this study, we developed two multiplex microsatellite polymerase chain reaction systems that consisted of 12 microsatellite markers (seven in system A and five in system B) for parentage assignment of orange‐spotted groupers. The theoretical exclusion probability per locus ranged from 0.079 to 0.436 when both parents were unknown (E‐1P) and from 0.191 to 0.614 when one parent was known (E‐2P). The combined exclusion probability values of 12 loci were 98.8% (E‐1P) and 100% (E‐2P), respectively. The accuracy of the parentage test for a mixed family (15 dams and 12 sires, 226 offspring) was 100%. In addition, two wild populations (79 individuals) were added as interferential offspring, only four of which were misassigned to the stock. The success rate of assignment was 98.69%. We provide a reliable microsatellite‐based assay for parentage assignment in orange‐spotted groupers, which will be useful for selective breeding and stock enhancement programs for this species and maybe other groupers.