scholarly journals A Multiplex Snapback Primer System for the Enrichment and Detection ofJAK2V617F andMPLW515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Zhiyuan Wu ◽  
Yunqing Zhang ◽  
Xinju Zhang ◽  
Xiao Xu ◽  
Zhihua Kang ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Amplification Efficiency ◽  
Primer Sets ◽  
Low Ionic Strength ◽  
Multiplex System

A multiplex snapback primer system was developed for the simultaneous detection ofJAK2V617F andMPLW515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets forJAK2andMPLmutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.

Multiplex Detection of Rotavirus A, B, C, Norovirus GI and GII, Adenovirus, Astrovirus and Sapovirus in a Single PCR System in Stool Samples From Children With Acute Diarrhea

2021 ◽  
Author(s):  
Wei Li ◽  
Wei-wei Li ◽  
Lin Li ◽  
Lin He ◽  
Wen-qing Xiang ◽  
...  
Keyword(s):  
Acute Diarrhea ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Cross Reaction ◽  
Melting Curve Analysis ◽  
Clinical Test ◽  
Rotavirus A ◽  
Diarrhea Virus ◽  
Intestinal Pathogens ◽  
Stool Samples

Abstract We developed a RT-PCR combined with melting curve analysis (RRCMC) method for simultaneous detection of rotavirus A, B, C, norovirus GI and GII, adenovirus, astrovirus and sapovirus. Stool samples were collected from 160 children with acute diarrhea and tested by RRCMC assay. A total of 71 patients were tested positive with norovirus, adenovirus or rotavirus. The RRCMC assay has high specificity. There is no internal cross-reaction through the 8 diarrhea viruses and no cross-reaction of other commonly intestinal pathogens and human genome. The detection limit was ranging from 1×102 to 1×105 copies/ml for each diarrhea virus. In conclusion, the RRCMC method is a suitable rapid clinical test for infectious viruses, with the advantages of high-throughput, low cost, high sensitivity and specificity.

Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization

2017 ◽  
Vol 52 (2) ◽  
pp. 186-195 ◽  
Author(s):  
Hsiang-Yun Tung ◽  
Wei-Chen Chen ◽  
Bor-Rung Ou ◽  
Jan-Ying Yeh ◽  
Yeong-Hsiang Cheng ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Single Infection ◽  
Pcr Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Viral Pathogens ◽  
Single Target ◽  
Primer Sets ◽  
Multiple Pathogens

Traditional serological enzyme-linked immunosorbent assay (ELISA) is routinely used to monitor pathogens during quarantine in most animal facilities to prevent possible infection. However, the ELISA platform is a single-target assay, and screening all targeted pathogens is time-consuming and laborious. In this study, to increase sensitivity and to reduce diagnosis time for high-throughput processes, multiplex PCR and DNA biochip techniques were combined to develop a multi-pathogen diagnostic method for use instead of routine ELISA. Eight primer sets were designed for multiplex PCR to detect genes from seven targeted bacterial and viral pathogens. DNA–DNA hybridization was conducted on a biochip following the multiple PCR analysis. Using this method, a total of 24 clinical samples were tested, and the result showed that not only single infection but also co-infection by multi-pathogens can be detected. In conclusion, multiplex PCR coupled with a DNA biochip is an efficient method for detecting multi-pathogens in a reaction. This platform is a useful tool for quarantine services and disease prevention in animal facilities.

Detection of SYT-SSX Rearrangements in Synovial Sarcomas by Real-Time One-Step RT-PCR

2005 ◽  
Vol 8 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Marina N. Nikiforova ◽  
Pamela Groen ◽  
George Mutema ◽  
Yuri E. Nikiforov ◽  
David Witte
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Soft Tissue Sarcomas ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Simple Method ◽  
Synovial Sarcomas ◽  
One Step

Synovial sarcomas are aggressive tumors of adolescent and young adults that account for up to 10% of soft tissue sarcomas. Cytogenetically, they are characterized by translocation t(X;18), which is found in more than 95% of tumors. In most cases, it results in fusion of the SYT gene with the SSX1 or SSX2 gene, thus creating SYT-SSX1 or SYT-SSX2 rearrangement. The 2 types of gene fusion have been correlated with histologic variants and prognosis of synovial sarcomas. In this study, we developed a simple and rapid method for the simultaneous detection of SYT-SSX1 and SYT-SSX2 rearrangements by using a LightCycler real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) technology (Roche). Oligonucleotide probes were designed so that the donor probe would span a fusion point and the acceptor probe would be complementary to the SSX1 sequence but have 2 nucleotide mismatches with SSX2 sequence. Such a design allows simultaneous amplification of 2 types of rearrangement in the same reaction but distinguishes them based on differences in melting temperature detected by melting curve analysis after PCR. With this method, 27 tumors (9 synovial sarcomas and 18 nonsynovial sarcomas) were studied and showed SYT-SSX1 rearrangement in 6 cases and SYT-SSX2 in 3 cases. These results had complete correlation with the finding of conventional RT-PCR and direct sequencing. In conclusion, we have developed a fast, accurate, and simple method for the detection of 2 major types of SYT-SSX rearrangement by using LightCycler RT-PCR and melting curve analysis.

scholarly journals Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

2008 ◽  
Vol 54 (6) ◽  
pp. 964-972 ◽  
Author(s):  
Clara R L Teo ◽  
Wen Wang ◽  
Hai Yang Law ◽  
Caroline G Lee ◽  
Samuel S Chong
Keyword(s):  
Huntington Disease ◽  
Trinucleotide Repeat ◽  
Neurodegenerative Disorder ◽  
False Positive Rate ◽  
Screening Method ◽  
Melting Curve ◽  
Single Step ◽  
Cag Repeat ◽  
Clinical Samples ◽  
Melting Curve Analysis

Abstract Background: Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG trinucleotide repeat in exon 1 of the HTT (huntingtin) gene and typically has an adult onset. Molecular diagnosis and screening for HD currently involve separate amplification and detection steps. Methods: We evaluated a novel, rapid microplate-based screening method for HD that combines the amplification and detection procedures in a single-step, closed-tube format. We carried out both the PCR for the HTT CAG-repeat region and the subsequent automated melting-curve analysis of the amplicon in the same wells on the plate. To establish cutoff melting temperatures (Tms) for each allelic class, we used a panel of reference DNA samples of known CAG-repeat sizes that represent a range of HTT alleles [normal (≤26 repeats), intermediate (27–35 repeats), reduced penetrance expanded (36–39 repeats), and fully penetrant expanded (≥40 repeats)]. We also measured well-to-well variation in Tm across the thermal block and validated cutoff Tms with DNA samples from 5 different populations. We also conducted a blinded validation analysis of clinical samples from an additional 40 HD-affected and 30 unaffected individuals. Results: We observed a strong correlation between CAG-repeat size and amplicon Tm among the reference DNA samples. Use of the Tm cutoffs we established revealed that 5 samples from unaffected individuals had been misclassified as affected (1.1% false-positive rate). All samples from HD-affected and unaffected individuals were correctly identified in the blinded analysis. Conclusions: This simple and scalable homogeneous assay may serve as a convenient, rapid, and accurate screen to detect the presence of pathologic expanded HD alleles in symptomatic patients.

Prothrombin 20209C>T: Thirteen New Cases and Association with the 19911A>G Polymorphism.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1827-1827
Author(s):  
Ilka Warshawsky ◽  
Vinit Makkar ◽  
Kandice Kottke-Marchant
Keyword(s):  
Pulmonary Embolism ◽  
Venous Thrombosis ◽  
African Descent ◽  
Melting Curve ◽  
Restriction Enzymes ◽  
Curve Analysis ◽  
Routine Screening ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Hybridization Probe

Abstract During routine screening for the prothrombin 20210G&gt;A mutation as a risk factor for venous thrombosis, we and others have reported cases of prothrombin 20209C&gt;T heterozygotes that are enriched in individuals of non-Caucasian descent who have had thrombotic events and/or obstetrical complications. The prothrombin 20209C&gt;T variant, as well as six other variants close to position 20210, have been found by melting curve analysis using the LightCycler Instrument (Roche Molecular Biochemicals) and hybridization probes. Unlike assays which use restriction enzymes or allele-specific PCR, melting curve analysis can distinguish multiple base changes that lie within the region of a hybridization probe as long as the base change causes a significant change in melting temperature from expected results. We describe thirteen new prothrombin 20209C&gt;T heterozygous cases from unrelated individuals and one family study which were found during routine screening of clinical samples for the prothrombin 20210G&gt;A mutation. Ethnic and clinical information were available in 11/13 cases: Ten individuals were of African descent while one was Hispanic. Eight individuals had thrombotic events (venous thrombosis, pulmonary embolism, and/or stroke), one had a pregnancy loss, one had bilateral below the knee amputations due to severe peripheral vascular disease, and one had a questionable pulmonary embolism. We next examined the prevalence of the prothrombin 19911A&gt;G polymorphism in 20209C&gt;T heterozygotes since alleles 19911A and 20210A are in full linkage and since the 19911G allele has been associated with increases in both plasma prothrombin activity and venous thrombosis risk. Of twenty 20209C&gt;T heterozygotes, eighteen were 19911GG homozygous and two were 19911AG heterozygous. These results are consistent with linkage of alleles 20209T and 19911G and contrasts with the linkage of alleles 20210A and 19911A. Since the 19911G allele was found in all 20209C&gt;T patients, it is possible that the 19911G-20209T haplotype increases the risk for thrombosis and/or obstetric complications, especially among individuals of African descent.

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