scholarly journals Simple and rapid detection of common fetal aneuploidies using peptide nucleic acid probe-based real-time polymerase chain reaction

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Subeen Hong ◽  
Seung Mi Lee ◽  
Sohee Oh ◽  
So Yeon Kim ◽  
Young Mi Jung ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Peptide Nucleic Acid ◽  
Melting Curve ◽  
Peak Height ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

AbstractTo examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies. Using amniotic fluid samples, PNA probe based real-time PCR (Patio DEP Detection Kit; SeaSun Biomaterials, Korea) assay was performed. PNA probe was designed to hybridize to similar sequences located on different segments of target chromosomes (21, 18, and 13) and a reference chromosome. Amplification of target sequences and melting curve analysis was performed. When analyzing the melting curve, the ratio of the peak height of the target and reference chromosome was calculated and determined as aneuploidy if the ratio of peak height was abnormal. All the results from the PNA probe-based real-time PCR and melting curve analyses were compared to those from conventional karyotyping. Forty-two cases with common aneuploidies (24 of trisomy 21, 12 of trisomy 18, and 6 of trisomy 13) and 131 cases with normal karyotype were analyzed. When comparing the karyotyping results, the sensitivity and specificity of the PNA probe-based real-time PCR assay were both 100%. The level of agreement was almost perfect (k = 1.00). PNA real-time PCR assay is a rapid and easy method for detecting common aneuploidies.

DETECTION OF NOTCH1 c.7544_7545delCT MUTATION IN CHRONIC LYMPHOCYTIC LEUKEMIA USING CONVENTIONAL AND REAL-TIME POLYMERASE CHAIN REACTION

2016 ◽  
Vol 38 (2) ◽  
pp. 112-116 ◽  
Author(s):  
N I Bilous ◽  
I V Abramenko ◽  
A A Chumak ◽  
I S Dyagil ◽  
Z V Martina
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Lymphocytic Leukemia ◽  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Lymphocytic Leukemia ◽  
Melting Curve Analysis ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Polymerase Chain

Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted to real-time PCR. Results: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-consuming, showed advantage in the assessment of the amplification’s specificity (using the melting curve analysis). It also allows the quantitative assessment of NOTCH1-mutated clone. Conclusion: NOTCH1 c.7544_7545delCT mutation resulting in removal of the C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients.

Early and simultaneous detection ofNosema bombycis(Microsporidia: Nosematidae), nucleopolyhedrovirus (Baculoviridae), and densovirus (Parvoviridae) by multiplex real-time polymerase chain reaction inBombyx mori(Lepidoptera: Bombycidae)

2017 ◽  
Vol 149 (2) ◽  
pp. 265-275
Author(s):  
Shan Wu ◽  
Yong-Qiang He ◽  
Xing-Meng Lu ◽  
Xiao-Feng Zhang ◽  
Jiang-Bing Shuai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Early Detection ◽  
Bombyx Mori ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Post Inoculation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

AbstractAn effective multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of three major pathogens,Nosema bombycisNägeli (Microsporidia: Nosematidae),Bombyx morinucleopolyhedrovirus (Baculoviridae: genusAlphabaculovirus) (NPV), andBombyx moridensovirus (Parvoviridae: genusIteravirus) (DNV), in silkworms (Bombyx mori(Linnaeus); Lepidoptera: Bombycidae) was developed in this study. Polymerase chain reaction and real-time PCR tests and basic local alignment search tool searches revealed that the primers and probes used in this study had high specificities for their target species. The ability of each primer/probe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 8.5×103copies forN. bombycisandBombyx moriNPV (BmNPV) and 8.5×104copies forBombyx moriDNV (BmDNV). When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity, and high-throughput capabilities. Under the optimal volume ratio for the three primer/probe sets (3:2:2=N. bombycis:BmNPV:BmDNV), the multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation using DNA templates of the three pathogens in various combinations from individually infected silkworms; the early detection ofN. bombyciswas possible by day 3 post inoculation using the DNA isolated from the midgut ofN. bombycis-infected silkworms.

Optimization of 6-carboxy-X-rhodamine concentration for real-time polymerase chain reaction using molecular beacon chemistry

2007 ◽  
Vol 53 (3) ◽  
pp. 391-397 ◽  
Author(s):  
Gehua Wang ◽  
Erin Becker ◽  
Christine Mesa
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Quantitative Polymerase Chain Reaction ◽  
Luxs Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Shiga Toxin 2 ◽  
Polymerase Chain

The optimal 6-carboxy-X-rhodamine (ROX) concentration, which is used as a passive reference dye for real-time quantitative polymerase chain reaction (PCR) with molecular beacon chemistry, was determined with the Mx4000™ Multiplex Quantitative PCR System. Additionally, the effects of changing ROX concentrations on PCR reproducibility, Ct values, and efficiency were investigated with this system by using the PCR data obtained from amplification of the Escherichia coli shiga toxin 2 (stx2) gene and the Campylobacter jejuni luxS gene. This study indicated that different ROX concentrations influence many aspects of the real-time PCR reaction. ROX concentration variation could have consequences in the analysis of quantitative data and may lead to erroneous results. This study further indicated that the optimal ROX concentration is 60 nmol/L for real-time PCR, using molecular beacon chemistry for PCR assay of luxS and stx2 genes.

scholarly journals Early Detection of Leifsonia xyli subsp. xyli in Sugarcane Leaves by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 430-434 ◽  
Author(s):  
M. P. Grisham ◽  
Y.-B. Pan ◽  
E. P. Richard
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Close Agreement ◽  
Leaf Tissue ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Pcr Assays

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

scholarly journals Detection of the Janus kinase 2 V617F mutation using a locked nucleic-acid, real-time polymerase chain reaction assay

2018 ◽  
Vol 7 (1) ◽  
Author(s):  
Tshiphiri Senamela ◽  
Marleen Kock ◽  
Piet Becker ◽  
Joachim J.C. Potgieter
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Jak2 V617f ◽  
Locked Nucleic Acid ◽  
Jak2 V617f Mutation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
V617f Mutation

The purpose of this study was to develop a real time polymerase chain reaction (PCR) assay for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Sanger sequencing and a newly developed locked nucleic-acid, real-time PCR assay were used to detect the JAK2 V617F mutation. There was 100% agreement between the sequencing and PCR analysis. Both assays were able to detect the mutation in all 24 of the 60 test specimens harbouring the mutation.

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