scholarly journals Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

2017 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Ery Nourika Alfiraza ◽  
Esti Enjelina ◽  
Deni Pranowo
Keyword(s):  
Real Time ◽  
Annealing Temperature ◽  
Limit Of Detection ◽  
Melting Curve ◽  
High Specificity ◽  
Optimization Method ◽  
Amyl Alcohol ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Temperature Optimization

Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.

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 Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252887
Author(s):  
Renate Schneider ◽  
Aline Lamien-Meda ◽  
Herbert Auer ◽  
Ursula Wiedermann-Schmidt ◽  
Peter L. Chiodini ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Melting Curve ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Significant Rise ◽  
Quantitative Detection ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

scholarly journals MULTIPLEX SYBR GREEN ASSAY FOR CORONAVIRUS DETECTION USING FAST REAL-TIME RT-PCR

2020 ◽  
Vol 51 (2) ◽  
pp. 556-564
Author(s):  
Salah & et al.
Keyword(s):  
Respiratory Tract ◽  
Real Time ◽  
Melting Curve ◽  
First Record ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Upper Respiratory ◽  
Tract Infections

This study was aimed to provide a local database for detection of coronavirus (CoV) species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green reverse transcriptase real-time PCR (rRT-PCR) technique. A total of 500 samples was collected from individuals suffering from upper and/or lower respiratory tract diseases for testing of 4 CoV species (229E, OC43, NL63, and HKU1). RNA extracted, amplified and subsequent the positive samples sequencing. The results showed melting curve analysis (Tm) of the specific amplicons (79.73±0.36) and 9% positive for CoVs  and some of them have other co-infection such as influenza virus 26.67%, and TB 11.11%. On the other hands, the CoVs were detected 4.62% in upper respiratory samples and 20.39% with lower respiratory samples. Sequencing results pointed out two isolates were CoV-NL63 and four isolates were CoV-229E, with first record accession number MN086823.1 and MN086824.1, respectively in GenBank. In conclusion, this rRT-PCR showed the rapid and efficient detection of CoVs with few copies number. This allows being used for the diagnosis of CoVs along with other respiratory viruses in a multiplex assay to reduce processing time. Subsequent applied nested RT-PCR to overcome the low viral load.

scholarly journals Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Liuyang Hu ◽  
Bing Han ◽  
Qin Tong ◽  
Hui xiao ◽  
Donglin Cao
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Melting Curve ◽  
Cost Effective ◽  
Culture Method ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Fluorescence Melting Curve Analysis

Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods. A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results. Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions. Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

Detection of Multiple Bacterial Strains in Peritoneal Dialysis Patients based on Multiplex Real-time PCR with Melting Curve Analysis

2021 ◽  
Vol 19 (10) ◽  
pp. 127-131
Author(s):  
Dr. Faten Naeem Abbas ◽  
Amany Shakeir Jabber
Keyword(s):  
Peritoneal Dialysis ◽  
Real Time ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Bacterial Strains ◽  
Rt Pcr ◽  
Dialysis Patients ◽  
E Coli ◽  
Technique Failure

Peritoneal dialysis (PD) is an underutilized mode of dialysis therapy worldwide. Despite the reduction in peritonitis rate, peritonitis continues to be the main cause of technique failure. This study was undertaken to detection of the multiple bacterial strains in dialysis patients by melting curve analysis Real-time (RT-PCR). Ninety specimens of dialysate collected were blood culture and primers RT-PCR detection of the pathogens (bacterial). The results showed the most detected bacteria were Streptococcus pneumoniae (33%) followed by E. coli (31%), Staphylococcus aureus (13%), Klebsiella pneumoniae 11% then Pseudomonas aeruginosa (7%) respectively while the least frequency were recorded with Enterococcus faecalis with only (5%). From this study can concluded the identification of pathogens within two hours, Six bacterial pathogens have been detected and differentiated by using multiplex RT-PCR with peaks for melting curve study, high repeatability was exhibited for every amplification product.

scholarly journals Comparison of bluetongue virus detection and quantitation methods in south India

2014 ◽  
Vol 8 (10) ◽  
pp. 1307-1312
Author(s):  
Subhra Subhadra ◽  
Subrat Kumar ◽  
Veluvarthy VS Suryanarayana ◽  
Daggupati Sreenivasulu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Disease Diagnosis ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Animal Blood ◽  
Specificity And Sensitivity

Introduction: Bluetongue (BT), a vector-borne viral disease, primarily affects sheep. Of the 26 serotypes of BTV identified so far, 22 are reported to be circulating in India. Due to an increase in vector population and delays in disease diagnosis, the BT control program heavily relies on rapid and confirmatory diagnosis. Polymerase chain reaction (PCR)-based real-time detection assays may be an ideal method to detect the BTV genome in animal blood at an early stage of infection. Methodology: In this study, a SYBR green-based real-time RT-PCR assay was evaluated, validated, and compared with conventional RT-PCR. The specificity and sensitivity of an assay using BTV-2 RNA extracted from tenfold serially diluted (starting from 1.0 TCID50/mL) cell culture virus was also evaluated. Results: While conventional RT-PCR could detect 3.16×102 TCID50 of virus/mL, the real-time PCR test had a detection limit of 3.16×10-4 TCID50/mL. Melting curve analysis indicated the absence of non-specific amplification (R2 = 0.987). Out of the 32 infected blood samples examined, 24 tested positive for BTV RNA. Seven that were found negative through conventional PCR tested positive through real-time PCR. Conclusions: These results showed that the SYBR green-based real-time PCR assay is rapid, sensitive, and equally specific in the diagnosis of BT in BTV-affected animals.

scholarly journals Detecting Genes Expressed at Different Stages of Reproductive Arrest in Brassica oleracea

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 754C-754
Author(s):  
Denise V. Duclos* ◽  
Thomas Björkman
Keyword(s):  
Brassica Oleracea ◽  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Restriction Enzyme Digestion ◽  
Mads Box ◽  
Mads Box Genes ◽  
Rt Pcr ◽  
Inflorescence Meristem

Cauliflower (Brassica oleracea var. italica) and Broccoli (Brassica oleracea var. botrytis) differ mainly in the stage of reproductive arrest. Cauliflower curd is an inflorescence meristem, while broccoli arrests just before anthesis. Arabidopsis studies led to the hypothesis that a mutant BoCAL allele arrested cauliflower earlier. Later, a mutant in BoAP1 was found to have similar effects. These partially redundant genes, and several identified since, are present in multiple copies in B. oleracea. Understanding their role in the arrest requires quantification of transcript abundance analysis by real-time PCR. Designing selective PCR primers is a critical first step in the process. Designs were based on alignment among the genes of interest (MADS-box genes BoCAL, BoAP1, FUL, and the non MADS-box genes LFY and TFL1) and their paralogs. The high sequence similarity (some over 95%) makes the target transcripts difficult to distinguish. Therefore, primers were designed mostly for targets in the 3'UTR region in order to gain specificity. Short amplicons, 68bp to 200bp, were required for the high PCR efficiency required to quantify these low-abundance transcripts. Primers were evaluated by conventional RT-PCR and real-time PCR. By altering temperature, Total RNA was isolated from plants that were arrested at three developmental stages, inflorescence meristem (cauliflower), floral meristem (intermediate), and floral bud (broccoli) by varying temperature. RT-PCR products were single bands of the expected size, despite the high homology between genes under study. Real-time melting curve analysis (fluorescence derivative vs. melting temperature) corroborated the presence of a single amplicon. The identity of products was confirmed by sequencing and restriction enzyme digestion.

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