Insertion of amelogenin marker in the 38 Hid-Indels multiplex reaction

2015 ◽  
Vol 5 ◽  
pp. e165-e166
Author(s):  
D.F. Braganholi ◽  
F.S. Polverari ◽  
I.B. Ambrosio ◽  
R.M.B. Cicarelli
Keyword(s):  
Multiplex Reaction

Development of Two Multiplex Polymerase Chain Reactions for the Detection of Enterotoxigenic Strains of Staphylococcus aureus Isolated from Foods

2003 ◽  
Vol 66 (6) ◽  
pp. 1055-1062 ◽  
Author(s):  
GABRIELA NÁJERA-SÁNCHEZ ◽  
ROGELIO MALDONADO-RODRÍGUEZ ◽  
PATRICIA RUÍZ OLVERA ◽  
LYDIA MOTA de la GARZA
Keyword(s):  
Staphylococcus Aureus ◽  
Simultaneous Detection ◽  
Type A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Powdered Milk ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Type D ◽  
Multiplex Reaction ◽  
Polymerase Chain

Two multiplex polymerase chain reactions were developed for the detection of enterotoxigenic strains of Staphylococcus aureus: one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD). Both reactions were standardized with the use of the reference enterotoxigenic strains of S. aureus: FRI 722, producer of staphylococcal enterotoxin (SE) type A (SEA); FRI 1007, producer of SEB; FRI 137, producer of SEC1; FRI 472, producer of SED; and FRI 326, producer of SEE. Optimized methods were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products by the Baird-Parker technique. The enterotoxigenic capacities of the strains were determined by the indirect enzyme-linked immunosorbent assay (ELISA) with the use of reference staphylococcal toxins and antitoxins. Fifty of the 51 strains isolated were enterotoxigenic and produced one to four enterotoxin types, with the most frequently produced types being SEA and SED. Levels of correlation between the presence of genes that code for the production of SE (as determined by polymerase chain reaction) and the expression of these genes (as determined by the indirect ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB.

Genetic variability at nine STR loci in the Chueta (Majorcan Jews) and the Balearic populations investigated by a single multiplex reaction

2000 ◽  
Vol 113 (5) ◽  
pp. 263-267 ◽  
Author(s):  
C. Tomàs ◽  
A. Picornell ◽  
J. A. Castro ◽  
M. M. Ramon ◽  
L. Gusmão ◽  
...  
Keyword(s):  
Genetic Variability ◽  
Str Loci ◽  
Multiplex Reaction

Self-Driven Multiplex Reaction: Reactant and Product Diffusion via a Transpiration-Inspired Capillary

2021 ◽  
Author(s):  
Bingda Chen ◽  
Feifei Qin ◽  
Meng Su ◽  
Zeying Zhang ◽  
Qi Pan ◽  
...  
Keyword(s):  
Product Diffusion ◽  
Multiplex Reaction

scholarly journals Comparative Analysis of Verification Parameters of Event-Specific Methods in GMO Maize

2018 ◽  
Vol 2 (1) ◽  
pp. 8
Author(s):  
Anesa Ahatović ◽  
Adaleta Durmić-Pašić
Keyword(s):  
Limit Of Detection ◽  
Certified Reference Materials ◽  
Simultaneous Detection ◽  
Specific Marker ◽  
The European Union ◽  
Gm Crop ◽  
Gm Maize ◽  
Absolute Limit ◽  
Multiplex Reaction

The second most dominant genetically modified (GM) crop is maize. Increasing number of GM maize events puts significant pressure on GMO testing laboratories to achieve the level of competence necessary to fulfill legal requirements. In the European Union, Polymerase Chain Reaction (PCR) is the method of choice for identification and quantification of GMOs. We performed verification of validated methods for identification of four GM maize events. Additionaly we aimed to explore the option of designing a method for simultaneous detection of these events in a multiplex PCR reaction. DNA was extracted from certified reference materials (CRM) using validated CTAB extraction protocol. Concentration of DNA was measured using Qubit dsDNA Broad Range Assay. Amplification of taxon specific marker for maize and all event-specific methods was performed according to the JRC Compendium of Reference Methods for GMO Analysis. Absolute limit of detection (LODabs) was determined for taxon specific and four event specific RealTime PCR based methods. DNA extracted from CRMs showed sufficient concentration for downstream analyses and preparation of dilutions for determination of LODabs. Determined LODabs for all tested methods meet acceptance criteria. As expected, the methods performance with respect to the repeatability and precision decline with the decrease in concentration of the target. Event-specific GA21 and NK603 methods show high Ct values at the determined LODabs. However, by adjusting the concentrations of primers and probes sensitivity of these two methods should be improved. Considering that the amplicons for all five methods are quite short (<120 bp) optimization of multiplex reaction conditions for simultaneous amplification should be feasible

Improving genetic monitoring of the northern hairy-nosed wombat (Lasiorhinus krefftii)

2014 ◽  
Vol 62 (3) ◽  
pp. 246 ◽  
Author(s):  
Lauren C. White ◽  
Alan Horsup ◽  
Andrea C. Taylor ◽  
Jeremy J. Austin
Keyword(s):  
Microsatellite Markers ◽  
Dna Analysis ◽  
Genotype Frequencies ◽  
Abundance Estimates ◽  
Multiplex Reaction ◽  
Hardy Weinberg Equilibrium ◽  
Genetic Techniques ◽  
Polymerase Chain ◽  
Future Population

The endangered northern hairy-nosed wombat (Lasiorhinus krefftii) has been monitored via remote sampling and genetic techniques since 2000, thus avoiding the detrimental effects on the animals of trapping and increasing the precision of abundance estimates. The currently available dinucleotide microsatellite markers used for this task are prone to stutter and other polymerase chain reaction artefacts, making allele calling difficult, and requiring costly duplication to ensure accuracy. To remedy this we have developed eight new tri- and tetranucleotide microsatellite markers that reduce the problem of stutter in DNA analysis. These new markers, along with three of the existing markers (two microsatellites and the SRY gender marker) were optimised in a single multiplex reaction that will reduce the time and cost of future northern hairy-nosed wombat hair censuses. We tested this new multiplex on 277 non-invasively collected hairs. One locus was rejected due to null-allele issues. The remaining nine microsatellite loci had two or three alleles. Genotype frequencies in the sample of detected individuals did not differ significantly from Hardy–Weinberg equilibrium and there was no evidence of linkage disequilibrium. This new multiplex provides comparable power to distinguish individuals, fewer issues with stutter artefacts and a reduced time and cost of analysis. It will be useful for future population censuses and long-term monitoring of individuals once they have been scored in previously genotyped and assigned samples.

scholarly journals A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches

2000 ◽  
Vol 90 (10) ◽  
pp. 1137-1144 ◽  
Author(s):  
G. J. Vandemark ◽  
J. M. Kraft ◽  
R. C. Larsen ◽  
M. A. Gritsenko ◽  
W. L. Boge
Keyword(s):  
Dna Markers ◽  
Pcr Primers ◽  
Single Step ◽  
Aphanomyces Euteiches ◽  
Diagnostic Pcr ◽  
Trial Site ◽  
Pcr Product ◽  
Multiplex Reaction ◽  
Pcr Products ◽  
Time Required

Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72°C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.

scholarly journals DEVELOPMENT AND TESTING OF A LOW-COST INACTIVATION BUFFER THAT ALLOWS DIRECT SARS-COV-2 DETECTION IN SALIVA

2021 ◽  
Author(s):  
Brandon Bustos-Garcia ◽  
Sylvia Garza-Manero ◽  
Nallely Cano-Dominguez ◽  
Dulce Maria Lopez Sanchez ◽  
Gonzalo Salgado-Montes de Oca ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Heat Inactivation ◽  
Independent Manner ◽  
Resource Limited ◽  
Multiplex Reaction ◽  
The Us ◽  
Diagnostic Standards ◽  
Control And Prevention ◽  
Commercial Kits

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places where good vaccination coverage has been achieved. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing implementation in resource-limited settings. It is therefore important to develop testing protocols that facilitate implementation and are inexpensive, fast, and sufficiently sensitive. In this work, we optimized the composition of a buffer (PKTP) containing a protease, a detergent, and an RNase inhibitor, that is compatible with the RT-qPCR chemistry, allowing for direct testing of SARS-CoV-2 from saliva in an RNA extraction-independent manner. This buffer is compatible with heat-inactivation reducing the biohazard risk of handling the samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (82.2% for the N1 and 84.4% for the N2 target, respectively) and correlations (R=0.77, p<0.001 for N1, and R=0.78, p<0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to reduce the number of PCR reactions per patient and further reduce overall costs while maintaining diagnostic standards in favor of massive testing.

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay

2016 ◽  
Vol 99 (4) ◽  
pp. 1038-1042
Author(s):  
Katie L Korchinski ◽  
Adrian D Land ◽  
David L Craft ◽  
Jennifer L Brzezinski
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Positive Control ◽  
Specific Gene ◽  
Gene Target ◽  
Tobacco Products ◽  
Chewing Tobacco ◽  
Multiplex Reaction ◽  
Internal Positive Control ◽  
Pcr Method

Abstract Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products—including chewing tobacco, pipe tobacco, and snuff—or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Sign in / Sign up

Export Citation Format

Share Document