Toward rapid testing for molecular authentication: Novel method for multianalyte identification of olive, sunflower, soy, sesame and corn DNA by visual biosensing.

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

The accurate and rapid identification of bacteria in the enteric tract is necessary for early treatment. In this study, we describeD a novel system which consists of a multiplex polymerase chain reaction (PCR) to simultaneously identify a group of six Enterobacteriaceae members including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter spp., Enterobacter cloacae and Salmonella typhi. Genus and species specific primers were designed for this group of pathogens and conventional multiplex PCR and SYBR green based real time PCR assays were performed to detect these pathogens. All the samples were analysed with a eubacterial real-time PCR assay that enables detection of bacterial DNA and then detection of the organisms was determined using genus and species specific PCR assays. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. Their PCR results matched with the conventional culture identifications. The conventional and SYBR green real time multiplex PCR assays takes only 3 h to be performed and has the potential to replace the conventional culture technique and thus can speed up the treatment process. This technique has the potential to be a valuable diagnostic tool for simultaneous identification of E. coli, K. pneumoniae, P. mirabilis, Citrobacter spp., E. cloacae and S. typhi.

BackgroundDetection of Tuberculosis agent like nontuberculous mycobacteria (NTM) species by culture and microscopic methods remains difficult and time consuming. A fast and reliable diagnosis of tuberculosis would greatly improve the control of the disease. The purpose of this study is to compare the conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay to culture method in terms of mycobacteria species detection.FindingsAmong the 117 positively cultured samples, nontuberculous mycobacteria (NTM) species were found in 9 samples of multiplex PCR reverse cross blot hybridization assay; compared to only 3 NTM species found in our conventional multiplex PCR, and 13 NTM species were successfully identified among 162 negatively cultured samples compared to only 5 NTM species identification in conventional multiplex PCR results.ConclusionsThe sensitivity of the multiplex PCR reverse cross blot hybridization assay comparing to culture method was 86.03%, the specificity is 35.46%, the positive predictive value was 41.94% and the negative predictive value was 82.41%. For conventional multiplex PCR these values are 81.62%, 38.65%, 41.89%, 79.51%, respectively. Furthermore, in terms of mycobacteria species detection, the conventional multiplex PCR was relatively equal compared to the multiplex PCR reverse cross blot hybridization assay, and to be particularly having no significant discrepant results on the identification of Mycobacteria tuberculosis in both methods.

A Rare Mutation in the Primer Binding Region of the Amelogenin Gene Can Interfere with Gender Identification

Identification of factor IX gene defects using a multiplex PCR and CSGE strategy—a first report

Microsphere Bead Arrays and Sequence Validation of 5/7/9T Genotypes for Multiplex Screening of Cystic Fibrosis Polymorphisms

Objective To learn about the genetic diversity of X-STR and to collect data of population genetics, the six X-STR loci were tested. Methods DXS6801、 DXS9902、 DXS6809、 DXS6803、 DXS6804 and DXS6799 were amplified in a single PCR reaction. PCR products were analyzed using capillary electrophoresis and ABI prism 3100 Genetic Analyzer, with GeneMapper ID 3. 1 Analysis Software. Results Allele typing with the systems was successful for all of six loci. When 202 unrelated male and 62 unrelated female individuals from Guangdong Han population were tested, 8,6, 11, 10,6 and 9 alleles were detected for DXS6801, DXS9902, DXS6809, DXS6803, DXS6804 and DXS6799, respectively. Polymorphism information content is 0. 662 3～0. 837 6. Power of discrimination in females is 0. 824 2～0. 951 1. Mean exclusion chance for X-STR in standard trios with daughters is 0. 594 9～0. 817 4. Conclusion The six loci in the multiplex system provide high polymorphism information for forensic identification and paternity testing, particularly for difficult paternity deficiency cases Key words: X-STR; Fluorescent multiplex PCR; Genetic polymorphism; Guangdong Han populationb

Background and Objective: Short Tandem Repeats (STR) show considerable differences among individuals in the population from which they used for identification. There are various methods for analysis of these STR loci, and capillary electrophoresis method already used as an international standard. Due to the high costs of this process, this study aimed to set up a Multiplex PCR method in some standard STR loci so that we can use its PCR product in STR analysis with different methods of HPLC, GC-Mass, and Capillary Electrophoresis.Materials and Methods: 8 typical STR loci in the identification selected according to their size in the two groups of four (CSF1PO, VWA, D18S51, PentaD and TPOX, Amelogenin, FGA, SE33) from NIST (National Institute of Standards and Technology). The above SSR primers prepared from Genbank and Monoplex PCR was designed based on their size. Then, with the changes in temperature conditions, magnesium ion, primers concentration, and setting-up, Hot Start Multiplex PCR of four markers was carried out. PCR product investigated on the agarose gel electrophoresis (3%) and the results of genotyping analyzed by Genetic Analyzer.Results: The Results showed that all STR loci under study are detectable as Monoplex PCR at a temperature of 62°-66° and 1.5 mM magnesium ion. Moreover, Multiplex PCR results showed that when the concentration of primer and temperature measured by the fixed concentration of magnesium, CSF1PO, and D18S51 loci bands are weaker than desired. Using a standard buffer and set Magnesium conditions against changes in the primer concentration and temperature, when Taq polymerase enzyme is added to test tubes at a temperature of 94°, Multiplex PCR bands are visible desirably. Capillary electrophoresis genotyping results obtained in all eight loci and the Locus FGA had the most allelic diversity and the loci TPOX and CSF1PO had the lowest allelic diversity. TPOX and CSF1PO loci had the lowest allelic frequencies, and FGA locus had the most allelic frequency. Moreover, about the determination of statistical indicators of identification using PowerStats V12 software, CSF1PO locus allocated the most RMP (0.219) and FGA locus the highest heterozygosity (100%) and the highest polymorphic rate (PIC) (0.82).Conclusion: The setup performed in this study showed that with two-step multiplex PCR procedure of four markers, PCR can be carried out for eight loci without additional real-time products that this shows proper conditions that we can use their PCR product in analyzing SRTs with different methods of HPLC, GC-Mass, and Capillary Electrophoresis. Besides, the FGA locus was raised as the best loci for identification in the study population concerning the high PD index and high polymorphism.

To apply capillary electrophoresis for rapid screening for Y microdeletions A set of 25 specific sequence tagged sites that cover the azoospermia factor a, b, and c regions of the Y chromosome was amplified in 5 fluorescent multiplex sets each including 5 primer pairs. One of the primers of each pair was labeled with a fluorescent tag attached to the 5'-end. After PCR amplification, analysis of the obtained PCR products was performed using capillary electrophoresis (ABI Prism 3100 Genetic Analyzer). The method was employed to determine Y microdeletions in azoospermic (n = 49) and severe oligozoospermic (n = 149) men. The number of PCR cycle (from 45 to 30) and the amount of DNA template (20-fold) used in fluorescent multiplex PCR were reduced because of the high sensitivity of capillary electrophoresis. Approximately 1000 multiplex PCR sets from 198 patients were analyzed simultaneously within 50 h. Y microdeletions were found in 3 out of the 198 azoospermic or severe oligozoospermic men. Application of capillary electrophoresis for detection of PCR products provides a semiautomated, high throughput method for rapid screening for microdeletions on the Y chromosome.

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.

Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.

McNemar’s test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR) assays confirmed the presence of the aggR gene (69%), ipaH gene (23%) and the stx gene (15%) carried by Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Enterohermorrhagic E. coli (EHEC), respectively, in river water samples collected from the Berg River (Paarl, South Africa). Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa). In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC) in 69%, stx (EHEC) in 15%, ipaH (EIEC) in 31% and eae (EPEC) in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC) was detected in 46% of the samples, while eae (EPEC) was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05) between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar’s test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health.

Comparison of Gold Standard Genescan with NGS-Based TCR-Beta Clonality Analysis Using Oncomine TCR Beta-Short Read Assay

Donkey meat is a rich source of protein excellent in indispensable amino acid and low in fat. Its quality has received great attention due to its high nutritional and medicinal value to human beings. Due to donkey resource scarcity and gradually increasing market demand for donkey meat products, adulterated donkey meat products with other low cost animal meat, especially sheep, cattle, pig and horse, were often found in market and had raised widespread concern in recent years. In this study, total 300 mg samples of binary mixtures, containing 90%, 50%, 25%, 10%, 5%, 1% (w/w) of donkey in horse meat respectively, were used for DNA extraction. Both conventional multiplex and real-time PCR techniques were developed for qualitatively and quantitatively detecting the adulteration in donkey meat. The multiplex PCR allowed rapid and simultaneous qualitative detection of donkey and four common adulterated species including sheep, cattle, pig and horse. Moreover, a normalized real-time PCR assay was further developed to detect and quantify the donkey mitochondrial DNA, and the results of simulate adulteration suggested this method could quantitatively detect donkey meat in range of 1–90% with high trueness. The results illustrated that both methods might be applied to detect commercial foodstuff adulteration. Therefore, both improved conventional multiplex and real-time PCR methods were developed to achieve qualitative and quantitative detection of the adulteration in donkey meat products.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right