scholarly journals Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6696 ◽  
Author(s):  
Pengpeng Yang ◽  
Yuzhu Song ◽  
Xueshan Xia ◽  
A-Mei Zhang
Keyword(s):  
Quantitative Pcr ◽  
Pcr Primers ◽  
Drug Resistant ◽  
First Line ◽  
Real Time Quantitative Pcr ◽  
Resistant Genes ◽  
Copy Numbers ◽  
Specificity And Sensitivity ◽  
Allele Specific ◽  
Line Drug

Tuberculosis (TB) is a worldwide health, economic, and social burden, especially in developing countries. Drug-resistant TB is the most serious type of this burden. Thus, it is necessary to screen drug-resistant mutations by using a simple and rapid detection method. A total of 32 pairs of allele-specific PCR (AS-PCR) primers were designed to screen mutation and/or wild-type alleles of 16 variations in four first-line drug-resistant genes (katG, rpoB, rpsL, and embB) of TB strains. A pair of primers was designed to amplify 16S rRNA gene and to verify successful amplification. Subsequently, we tested the specificity and sensitivity of these AS-PCR primers. The optimized condition of these AS-PCR primers was first confirmed. All mutations could be screened in general AS-PCR, but only 13 of 16 variations were intuitively investigated by using real-time quantitative PCR (qPCR) and AS-PCR primers. The results of specificity assay suggested that the AS-PCR primers with mutation and/or wildtype alleles could successfully amplify the corresponding allele under optimized PCR conditions. The sensitivity of nine pairs of primers was 500 copy numbers, and the other seven pairs of primers could successfully amplify correct fragments with a template comprising 103 or 104 copy numbers template. An optimized AS-qPCR was established to screen drug-resistant mutations in TB strains with high specificity and sensitivity.

scholarly journals Determination of RhD Zygosity: Comparison of a Double Amplification Refractory Mutation System Approach and a Multiplex Real-Time Quantitative PCR Approach

2001 ◽  
Vol 47 (4) ◽  
pp. 667-672 ◽  
Author(s):  
Rossa W K Chiu ◽  
Michael F Murphy ◽  
Carrie Fidler ◽  
Benny C Y Zee ◽  
James S Wainscoat ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Gene Dosage ◽  
Amplification Refractory Mutation System ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
Quantitative Pcr Assay

Abstract Background: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. Methods: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, ΔCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. Results: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the ΔCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. Conclusion: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

scholarly journals A Molecular Approach to Identification and Profiling of First-Line-Drug-Resistant Mycobacteria from Sputum of Pulmonary Tuberculosis Patients: Table 1

2012 ◽  
Vol 50 (6) ◽  
pp. 2082-2084 ◽  
Author(s):  
Imran Hussain Chowdhury ◽  
Aditi Sen ◽  
Bojlul Bahar ◽  
Avijit Hazra ◽  
Urmita Chakraborty ◽  
...  
Keyword(s):  
Pulmonary Tuberculosis ◽  
Drug Resistant ◽  
Molecular Approach ◽  
First Line ◽  
Tuberculosis Patients ◽  
Line Drug

PHOX2B Is a Novel and Specific Marker for Minimal Residual Disease Testing in Neuroblastoma

2008 ◽  
Vol 26 (33) ◽  
pp. 5443-5449 ◽  
Author(s):  
Janine Stutterheim ◽  
Annemieke Gerritsen ◽  
Lily Zappeij-Kannegieter ◽  
Ilona Kleijn ◽  
Rob Dee ◽  
...  
Keyword(s):  
Real Time ◽  
Minimal Residual Disease ◽  
Quantitative Pcr ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Specific Marker ◽  
Real Time Quantitative Pcr ◽  
Normal Tissues ◽  
Specificity And Sensitivity ◽  
Minimal Residual

Purpose Polymerase chain reaction (PCR)–based detection of minimal residual disease (MRD) in neuroblastoma can be used to monitor therapy response and to evaluate stem cell harvests. Commonly used PCR markers, tyrosine hydroxylase (TH) and GD2 synthase, have expression in normal tissues, thus limiting MRD detection. To identify a more specific MRD marker, we tested PHOX2B. Patients and Methods To determine PHOX2B, TH, and GD2 synthase expression in normal tissues, it was measured by real-time quantitative PCR in samples of normal bone marrow (BM; n = 51), peripheral blood (PB; n = 37), and peripheral-blood stem cells (PBSCs; n = 24). Then, 289 samples of 101 Dutch patients and 47 samples of 43 German patients were tested for PHOX2B and TH; these samples included 52 tumor, 214 BM, 32 BM, and 38 PBSC harvests. Of the 214 BM samples, 167 were compared with cytology, and 47 BM samples were compared with immunocytology (IC). Results In contrast to TH and GD2 synthase, PHOX2B was not expressed in any of the normal samples. In patient samples, PHOX2B was detected in 32% cytology-negative and in 14% IC-negative samples and in 94% of cytology-positive and in 90% of IC-positive BM samples. Overall, PHOX2B was positive in 43% compared with 31% for TH. In 24% of all samples, TH expression was inconclusive, which is similar to expression found in normal tissues. In 42% of these samples, PHOX2B expression was positive. Conclusion PHOX2B is superior to TH and GD2 synthase in specificity and sensitivity for MRD detection of neuroblastoma by using real-time quantitative PCR. We propose to include PHOX2B in additional prospective MRD studies in neuroblastoma alongside TH and other MRD markers.

scholarly journals Minimal Residual Disease Detection by Next-Generation Sequencing in Multiple Myeloma: A Comparison With Real-Time Quantitative PCR

2021 ◽  
Vol 10 ◽  
Author(s):  
Qiumei Yao ◽  
Yinlei Bai ◽  
Shaji Kumar ◽  
Elaine Au ◽  
Alberto Orfao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Next Generation Sequencing ◽  
Real Time ◽  
Quantitative Pcr ◽  
Residual Disease ◽  
Next Generation ◽  
Real Time Quantitative Pcr ◽  
Allele Specific ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Here we compared clonotype identification by allele-specific oligonucleotide real-time quantitative-PCR (ASO RQ-PCR) and next-generation sequencing (NGS) in 80 multiple myeloma patients. ASO RQ-PCR was applicable in 49/55 (89%) and NGS in 62/78 (80%). Clonotypes identified by both methods were identical in 33/35 (94%). Sensitivity of 10−5 was confirmed in 28/29 (96%) by NGS while sensitivity of RQ-PCR was 10−5 in 7 (24%), 5 × 10−5 in 15 (52%), and 10−4 in 7 (24%). Among 14 samples quantifiable by ASO RQ-PCR, NGS yielded comparable results in 12 (86%). Applicability of NGS can be improved if immunoglobulin heavy-chain incomplete DJ primers are included.

scholarly journals Comparison of Different Approaches To QuantifyStaphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

2001 ◽  
Vol 67 (7) ◽  
pp. 3122-3126 ◽  
Author(s):  
Ingeborg Hein ◽  
Angelika Lehner ◽  
Petra Rieck ◽  
Kurt Klein ◽  
Ernst Brandl ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Taqman Probe ◽  
Gene Copy ◽  
Real Time Quantitative Pcr ◽  
Copy Numbers ◽  
Gene Copies ◽  
Different Types ◽  
Pure Cultures ◽  
Gene Copy Numbers

ABSTRACT Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures ofS. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60nuc gene copies/μl) than using a fluorigenic TaqMan probe (6 nuc gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 � 102 to 6.4 � 102 copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.

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