Multiplex Polymerase Chain Reaction Panel Research Articles

Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels

We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.

Development of Genetic Markers for Triploid Verification of the Pacific Oyster, Crassostrea gigas.

The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.

Etiology and Seasonality of Viral Respiratory Infections in Rural Honduran Children

Limited data are available in Honduras that describe the etiology and seasonality of respiratory infections, especially in rural outpatient settings. Better data may lead to improved therapeutic and preventive strategies. The goal of our study was to determine the viral etiology and seasonality of acute respiratory infections in a rural Honduran population of children. Prospective clinic surveillance was conducted to identify children < 5 years of age presenting with respiratory symptoms of < 5 days duration. We obtained data on age, sex, medical history, breastfeeding history, symptoms, risk factors, household setting, temperature, respiratory rate and chest examination findings. To assess the association between specific viruses and weather, regional meteorological data were collected. Nasopharyngeal samples were tested for 16 respiratory viruses using a multiplex polymerase chain reaction panel. From February 2010 through June 2011, 345 children < 5 years of age were enrolled; 17%, 23%, 30% and 31% were <6, 6-11, 12-23 and 24-60 months old, respectively. Including all clinics in the region, 44.5% of patients < 5 years of age with documented respiratory diagnoses were enrolled. At least 1 virus was identified in 75.4% children, of which 7.5% were coinfections; 13.3% were positive for parainfluenza, 11.9% for influenza, 8.1% for human metapneumovirus and 7.5% for respiratory syncytial virus. Rainfall correlated with parainfluenza (P < 0.0001), influenza (P < 0.0001), human metapneumovirus (P = 0.0182) and respiratory syncytial virus (P < 0.0001). These results suggest that the spectrum of viruses in ill, rural, Honduran children is similar to that in North and Central America, although the seasonality is typical of some tropical regions.

Mucinous breast carcinomas lack PIK3CA and AKT1 mutations

Activating point mutations in the phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) are among the most common molecular defects in invasive breast cancer. Point mutations in the downstream kinase AKT1 are seen in a minority of carcinomas. These mutations are found preferentially in estrogen receptor-positive and Her2-positive breast carcinomas; however, special morphologic types of breast cancer have not been well studied. Twenty-nine cases of pure invasive mucinous carcinoma and 9 cases of ductal carcinoma with mucinous differentiation were screened for a panel of point mutations (>321 mutations in 30 genes) using a multiplex polymerase chain reaction panel with mass spectroscopy readout. In addition, associated ductal carcinoma in situ, hyperplasia, or columnar cell lesions were separately tested where available (25 lesions). In 3 invasive cases and 15 ductal carcinoma in situ/proliferative lesions, PIK3CA hotspot mutations were, instead, tested by direct sequencing. No point mutations were identified in invasive mucinous breast carcinoma. This contrasts with the 35% frequency of PIK3CA mutations in a comparative group of invasive ductal carcinomas of no special type. Interestingly, PIK3CA hotspot point mutations were identified in associated ductal carcinoma in situ (3/14) and hyperplasia (atypical ductal hyperplasia [2/3], usual ductal hyperplasia [2/3], columnar cell change [1/5]), suggesting that PIK3CA mutations may play a role in breast epithelial proliferation. This series represents the largest study, to date, of PIK3CA genotyping in mucinous carcinoma and supports the unique pathogenetics of invasive mucinous breast carcinoma.

Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Major Agents Causing Calf Diarrhea in Feces

Calf diarrhea is a major economic burden to the bovine industry. Since multiple infectious agents can be involved in calf diarrhea, and the detection of each of the causative agents by traditional methods is laborious and expensive, a panel of 2 multiplex real-time polymerase chain reaction (PCR) assays was developed for rapid and simultaneous detection of the 5 major bovine enteric pathogens (i.e., Bovine coronavirus [BCoV; formally known as Betacoronavirus 1], group A Bovine rotavirus [BRV], Salmonella spp., Escherichia coli K99(+), and Cryptosporidium parvum). The estimated detection limit (i.e., analytic sensitivity) of the panel was 0.1 TCID(50) (50% tissue culture infective dose) for BCoV and group A BRV; 5 and 0.5 colony-forming units for E. coli K99(+) and Salmonella, respectively; and 50 oocysts for Cryptosporidium per reaction. In testing 243 fecal samples obtained from submissions to the Iowa State University Veterinary Diagnostic Laboratory or from experimental animals with known infection status, the newly developed multiplex real-time PCR panel simultaneously detected all 5 pathogens directly from fecal samples and was more rapid and sensitive than the traditional diagnostic tests. The PCR panel showed 89%-97% agreement with those conventional diagnostic tests, demonstrating diagnostic sensitivity equal to or better than that of the conventional tests. In conclusion, the multiplex real-time PCR panel can be a tool for a timely and accurate diagnosis of calf diarrhea associated with BCoV, group A BRV, E. coli K99(+), Salmonella, and/or Cryptosporidium.