Development of a rapid and reliable single-tube multiplex real-time PCR method for HLA-A*24:02 genotyping

2019 ◽  
Vol 20 (11) ◽  
pp. 803-812
Author(s):  
Yanxia Wang ◽  
Tingting Zhang ◽  
Lirong Zhang ◽  
Yanrui Pei ◽  
Lili Zhao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection Method ◽  
Qpcr Assay ◽  
Specific Primers ◽  
Single Tube ◽  
Taqman Probes ◽  
Positive Rate ◽  
Allele Specific ◽  
Pcr Method ◽  
Polymerase Chain

Aim: HLA-A*24:02 is significantly associated with cutaneous adverse drug reactions caused by aromatic antiepileptic drugs. Here, we aimed to establish a fast and reliable detection method for HLA-A*24:02 genotyping. Methods: A single-tube multiplex quantitative real-time polymerase chain reaction (qPCR) assay for HLA-A*24:02 genotyping was established by combining allele-specific primers with TaqMan probes. Results: A 100% concordance was observed between qPCR and SBT result in 106 Han subjects. The detection limit of the new method was 0.05 ng DNA. The positive rate of HLA-A*24:02 in Tibetans (55.6%, n = 81) was significantly higher than those in Han (34%, n = 106), Uighur (27.5%, n = 102), Bouyei (25.9%, n = 116) and Miao populations (26.5%, n = 113). Conclusion: The newly established qPCR assay was reliable for HLA-A*24:02 screening in clinical applications.

Interlaboratory Validation Study of an Event-Specific Real-time Polymerase Chain Reaction Detection Method for Genetically Modified 55-1 Papaya

2013 ◽  
Vol 96 (5) ◽  
pp. 1054-1058
Author(s):  
Akio Noguchi ◽  
Kosuke Nakamura ◽  
Kozue Sakata ◽  
Tomoko Kobayashi ◽  
Hiroshi Akiyama ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Genetically Modified ◽  
Pcr Analysis ◽  
Labeling Regulations ◽  
Interlaboratory Validation ◽  
Endogenous Reference ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Genetically modified (GM) papaya line 55-1 (55-1) is resistant to papaya ringspot virus infection, and is commercially available in several countries. A specific detection method for 55-1 is required for mandatory labeling regulations. An event-specific real-time PCR method was developed by our laboratory. To validate the method, interlaboratory validation of event-specific qualitative real-time PCR analysis for 55-1 was performed in collaboration with 12 laboratories. DNA extraction and real-time PCR reaction methods were evaluated using 12 blind samples: six non-GM papayas and six GM papayas in each laboratory. Genomic DNA was highly purified from all papayas using an ion-exchange column, and the resulting DNA sample was analyzed using real-time PCR. Papaya endogenous reference gene chymopapain (CHY) and the event-specific 55-1 targeted sequence were detected in GM papayas whereas CHY alone was detected in non-GM papayas in all laboratories. The cycle threshold values of CHY and the 55-1 targeted sequence showed high repeatability (RSDr 0.6–0.8%) and reproducibility (RSDR 2.2–3.6%). This study demonstrates that the 55-1 real-time PCR detection method is a useful and reliable method to monitor 55-1 papaya in foods.

A novel single-tube multiplex real-time PCR assay for genotyping of thiopurine intolerance-causing variant NUDT15 c.415C>T

2021 ◽  
pp. 153537022110265
Author(s):  
Xiaoyun Lian ◽  
Yanwei Li ◽  
Lan Li ◽  
Kaicheng U ◽  
Wenxia Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sanger Sequencing ◽  
Clinical Settings ◽  
Pcr Assay ◽  
Established Method ◽  
Single Tube ◽  
Taqman Probes ◽  
Autoimmune Conditions ◽  
Allele Specific

Thiopurines are commonly used in the treatment of acute lymphoblastic leukaemia and autoimmune conditions, can be limited by myelosuppression. The NUDT15 c.415C>T variant is strongly associated with thiopurine-induced myelosuppression, especially in Asians. The purpose of this study was to develop a fast and reliable genotyping method for NUDT15 c.415C>T and investigate the polymorphic distribution among different races in China. A single-tube multiplex real-time PCR assay for NUDT15 c.415C>T genotyping was established using allele-specific TaqMan probes. In 229 samples, the genotyping results obtained through the established method were completely concordant with those obtained by Sanger sequencing. The distributions of NUDT15 c.415C>T among 173 Han Chinese, 48 Miaos, 40 Kazakhs, and 40 Kirghiz were different, with allelic frequencies of 0.06, 0.02, 0.07, and 0, respectively. This method will provide a powerful tool for the implementation of the genotyping-based personalized prescription of thiopurines in clinical settings.

scholarly journals Peptide Nucleic Acid (PNA)-Enhanced Specificity of a Dual-Target Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Assay for the Detection and Differentiation of SARS-CoV-2 from Related Viruses

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 775
Author(s):  
Won-Suk Choi ◽  
Ju Hwan Jeong ◽  
Halcyon Dawn G. Nicolas ◽  
Sol Oh ◽  
Khristine Joy C. Antigua ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Peptide Nucleic Acid ◽  
Detection Method ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Dual Target

The threat posed by coronaviruses to human health has necessitated the development of a highly specific and sensitive viral detection method that could differentiate between the currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related coronaviruses (SARSr-CoVs). In this study, we developed a peptide nucleic acid (PNA)-based real-time quantitative polymerase chain reaction (RT-qPCR) assay targeting the N gene to efficiently discriminate SARS-CoV-2 from other SARSr-CoVs in human clinical samples. Without compromising the sensitivity, this method significantly enhanced the specificity of SARS-CoV-2 detection by 100-fold as compared to conventional RT-qPCR. In addition, we designed an RT-qPCR method for the sensitive and universal detection of ORF3ab-E genes of SARSr-CoV with a limit of detection (LOD) of 3.3 RNA copies per microliter. Thus, the developed assay serves as a confirmative dual-target detection method. Our PNA-mediated dual-target RT-qPCR assay can detect clinical SARS-CoV-2 samples in the range of 18.10–35.19 Ct values with an 82.6–100% detection rate. Furthermore, our assay showed no cross-reactions with other coronaviruses such as human coronaviruses (229E, NL63, and OC43) and Middle East respiratory syndrome coronavirus, influenza viruses (Type B, H1N1, H3N2, HPAI H5Nx, and H7N9), and other respiratory disease-causing viruses (MPV, RSV A, RSV B, PIV, AdV, and HRV). We, thus, developed a PNA-based RT-qPCR assay that differentiates emerging pathogens such as SARS-CoV-2 from closely related viruses such as SARSr-CoV and allows diagnosis of infections related to already identified or new coronavirus strains.

scholarly journals Gaeumannomyces graminis vars. avenae, graminis, and tritici Identified Using PCR Amplification of Avenacinase-like Genes

Plant Disease ◽  
2002 ◽  
Vol 86 (6) ◽  
pp. 652-660 ◽  
Author(s):  
Sansanalak Rachdawong ◽  
Carole L. Cramer ◽  
Elizabeth A. Grabau ◽  
Verlyn K. Stromberg ◽  
George H. Lacy ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rapid Identification ◽  
Gaeumannomyces Graminis ◽  
Specific Primers ◽  
Single Tube ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Cereal Fields ◽  
Take All

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiating among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers. Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5′ primers specific for Gga, Ggt, and Ggg and a single 3′ common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5′ primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.

scholarly journals Detection of pork in meatballs using probe TaqMan Real-time Polymerase Chain Reaction

Food Research ◽  
2020 ◽  
Vol 4 (5) ◽  
pp. 1563-1568
Author(s):  
S. Orbayinah ◽  
A. Hermawan ◽  
Sismindari ◽  
A. Rohman
Keyword(s):  
Polymerase Chain Reaction ◽  
Wild Boar ◽  
Real Time ◽  
Chain Reaction ◽  
Specific Primers ◽  
Taqman Probes ◽  
Mitochondrial Region ◽  
D Loop ◽  
Polymerase Chain ◽  
Species Specific

This study aimed to develop a TaqMan Real-Time Polymerase Chain Reaction method, using a novel primer for detection of pork adulteration in meatballs. The study is important as it described a TaqMan method for product adulteration analysis. TaqMan is known to have a more specific result compared to SYBR green analytical method. Assay in the study combined species-specific primers and TaqMan probes to targeting 153 bp fragment of D-loop mitochondrial region of pork. A specificity test was conducted on fresh tissues of pork, beef, chicken, wild boar, dog, and mouse. Meatballs as samples were prepared from a mixture of pork-beef and wild boar-beef with concentrations as follows: 5%, 10%, 25%, 75%, 90%, and 100%. The linearity and sensitivity of the method were performed by measuring the amplification curve from the dilution series, namely 1000, 200, 100, 10, 5, 1, and 0.5 pg/μL of DNA, extracted from 100% pork meatballs. A repeatability test was conducted as many as six repetitions on 100% pork and 100% wild boar meatballs. This study showed that mitochondrial D-loop species-specific primers and TaqMan probes could identify the DNA of pork and wild boar on the fresh tissues. Additionally, it also resulted in a threshold cycle (Ct) of 17.02 and 17.95 for pork, 22.22 for wild boar, while the negative result for others. The detection limit has shown 5 pg in the meatball formulation. The Relative Standard Deviation (RSD) of repeatability was 1.936% for pork, while 2.140% for wild boar. The developed method was also applied to analyzing commercial meatballs. A TaqMan real-time PCR analytical method using specific primer targeting on 153 bp fragment of the D-loop mitochondrial region could be applied as a standard method for identifying pork and wild boar in food samples intended for halal authentication studies

scholarly journals Real-Time Polymerase Chain Reaction Detection of Fishmeal in Feedstuffs

2010 ◽  
Vol 93 (6) ◽  
pp. 1768-1777 ◽  
Author(s):  
Irene Martí ◽  
Teresa García ◽  
Maria Rojas ◽  
Nicolette Pegels ◽  
Miguel ángel Pavón ◽  
...  
Keyword(s):  
Real Time ◽  
Ribosomal Rna ◽  
Animal Species ◽  
Positive Control ◽  
Specific Primers ◽  
Ribosomal Rna Gene ◽  
18S Ribosomal Rna Gene ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Eukaryotic Organisms

Abstract A SYBR&lt;sup/&gt; Green PCR system was developed for detection of fishmeal in feedstuffs. The real-time PCR method combines the use of fish-specific primers that amplify an 87 base pair (bp) fragment of the mitochondrial 12S ribosomal RNA gene from fish species, and a positive control primer pair that amplifies a 99 bp fragment of the nuclear 18S ribosomal RNA gene in all eukaryotic organisms. The specificity of the primers was tested against 52 animal species and six plant species. Reference feedstuff samples were successfully tested for the presence of fishmeal, demonstrating the applicability of the assay to feedstuffs.

scholarly journals Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250942
Author(s):  
Huseyin Tombuloglu ◽  
Hussein Sabit ◽  
Ebtesam Al-Suhaimi ◽  
Reem Al Jindan ◽  
Khaled R. Alkharsah
Keyword(s):  
Real Time ◽  
Single Gene ◽  
False Negative ◽  
Diagnostic System ◽  
Virus Detection ◽  
Current Method ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Positive Results ◽  
Good Agreement

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.

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