scholarly journals High-throughput variant detection using a color-mixing strategy in real-time PCR reactions

2021 ◽  
Author(s):  
Nina Guanyi Xie ◽  
Kerou Zhang ◽  
Ping Song ◽  
Renqiang Li ◽  
Junfeng Luo ◽  
...  
Keyword(s):  
Single Gene ◽  
Rpob Gene ◽  
Genetic Alterations ◽  
Resistance Mutations ◽  
Isoniazid Resistance ◽  
Tuberculosis Diagnosis ◽  
Variant Detection ◽  
Color Mixing ◽  
Multiple Variants ◽  
Gene Probing

Many diseases are related to multiple genetic alterations along a single gene. Probing for highly multiple (>10) variants in a single qPCR tube is not possible due to a limited number of fluorescence channels and one variant per channel, so many more tubes are needed. Here, we experimentally validate our novel color-mixing strategy that uses fluorescence combinations as digital color codes to probe multiple variants simultaneously. The color-mixing strategy relies on a simple intra-tube assay that can probe for 15 variants as part of an inter-tube assay that can probe for an exponentially increased number of variants. The color-mixing strategy is achieved using multiplex double-stranded toehold probes modified with fluorophores and quenchers; the probes are designed to be quenched or luminous after binding to wildtype or variant templates. We used the color-mixing strategy to probe for 21 pathogenic mutations in thalassemia and distinguishing between heterozygous and homozygous variants in 6 tubes, with a specificity of 99% and a sensitivity of 94%. To support tuberculosis diagnosis, we used the same strategy to simultaneously probe in Mycobacterium tuberculosis for rifampicin-resistance mutations occurring within one 81-bp region and one 48-bp region in rpoB gene, plus five isoniazid-resistance mutations in inhA and katG genes.

Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production

Cytokine ◽  
2022 ◽  
Vol 151 ◽  
pp. 155788
Author(s):  
Darya V. Urusova ◽  
Joseph A. Merriman ◽  
Ananya Gupta ◽  
Liang Chen ◽  
Barun Mathema ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Cytokine Production ◽  
Rpob Gene ◽  
Resistance Mutations ◽  
Host Macrophage ◽  
Rifampin Resistance ◽  
Macrophage Cytokine

scholarly journals Loss of PTEN in Fallopian Tube Epithelium Results in Multicellular Tumor Spheroid Formation and Metastasis to the Ovary

Cancers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 884 ◽  
Author(s):  
Matthew Dean ◽  
Vivian Jin ◽  
Tova M. Bergsten ◽  
Julia R. Austin ◽  
Daniel D. Lantvit ◽  
...  
Keyword(s):  
Fallopian Tube ◽  
Single Gene ◽  
Similar Rate ◽  
Genetic Alterations ◽  
Wild Type ◽  
Spheroid Formation ◽  
Multicellular Tumor Spheroid ◽  
Ovarian Stroma

High-grade serous ovarian cancer (HGSOC) can originate in the fallopian tube and then spread to the ovary. Our objective was to evaluate the role of multicellular tumor spheroids (MTS) in ovarian metastasis. By testing a panel of murine oviductal epithelial (MOE) cells with genetic alterations mimicking those seen in HGSOC, we found that loss of PTEN allowed MTS formation under ultra-low adhesion conditions. Confirming these results in vivo, MTS-like structures were observed in the oviducts of PAX8Cre/+ PTENflox/flox mice. MOE PTENshRNA cells could incorporate up to 25% wild type cells into MTS, while higher percentages of wild type cells resulted in a loss of MTS formation. MTS formation allowed MOE PTENshRNA cells to survive better under ultra-low adhesion conditions than control cells. MTS also attached to the ovarian stroma, as would be exposed during ovulation. Interestingly, MTS more robustly cleared monolayers of murine ovarian surface epithelia than murine ovarian fibroblasts. When xenografted into the ovarian bursa, OVCAR8 MTS were able to form tumors in the ovary at a similar rate as an equal number of OVCAR8 cells grown on traditional cell culture plastic. In conclusion, loss of a single gene (PTEN) allows the fallopian tube epithelia to form MTS, which survive better under ultra-low adhesion conditions, attach to the extracellular matrix exposed during ovulation, and colonize the ovary. These results suggest that MTS may contribute to seeding of the ovary in HGSOC patients.

scholarly journals Assessment of Gene-Xpert MTB RIF Program Implementation and the Challenges For Enhanced Tuberculosis Diagnosis in Nigeria

2016 ◽  
Vol 12 (2) ◽  
pp. 1-7 ◽  
Author(s):  
G Mustapha ◽  
O Jumoke ◽  
P Nwadike ◽  
E Emeka ◽  
G Akang ◽  
...  
Keyword(s):  
Program Implementation ◽  
Single Gene ◽  
Risk Groups ◽  
Health Facilities ◽  
Rifampicin Resistance ◽  
Public Health Response ◽  
Hiv Services ◽  
Challenges And Opportunities ◽  
Early Case ◽  
Tuberculosis Diagnosis

Introduction: Gene-Xpert MTBRIF, rapid tuberculosis and rifampicin resistance diagnostic technology is implemented in Nigeria to enhance public health response to tuberculosis diagnosis in HIV patients with presumed tuberculosis (TB), and presumed cases of drug resistant TB. The aim of the paper is to share experience on programmatic issues on Xpert MTB RIF roll-out.Methodology: Program implementation data from 22 Xpert laboratories for period between September 2011 and December 2013 were analyzed to evaluate outcomes and identify challenges and opportunities for strengthening tuberculosis detection in Nigeria.Results: A total of 12249 patients received single gene-Xpert test at 10 secondary (S), 10 Tertiary (T) and 2 private (P) health facilities over 25 months. The tests were valid in 10948 patients, and 3669/10948 (33.5%) were positive for Mycobacterium tuberculosis (MTB). In 815/3669 (22.2%) of the MTB cases, the bacteria were resistant to rifampicin. Rifampicin resistance was inconclusive (indeterminate) in 509/12249 (4.2%) while the test failed in 792/12249 (6.5%). The program was noticeably limited to health facilities above primary centers; there were prolonged delays in the diagnosis and treatment with limited on-site synergy between TB/HIV services. Reducing diagnostic delays and integrating TB/HIV services into the gene-Xpert program will enhance early case detection and enrollment for care in Nigeria.Conclusion: The model Gene-Xpert MTBRIF program implemented in Nigeria targets specific risk groups with high number of cases detected. Diagnoses of tuberculosis and resistance to rifampicin could be enhanced by offering integrated TB/HIV services; improving patient and sample flow/referral; proper documentation of test outcomes and alignment with DR-TB management.SAARC J TUBER LUNG DIS HIV/AIDS, 2015 XII (2), page: 1-7  

Expanded molecular routine testing for targetable mutations in non-small cell lung cancer to reveal frequent co-occuring mutations.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20596-e20596
Author(s):  
Anna Kostenko ◽  
Jana Fassunke ◽  
Susanne Steinhauser ◽  
Matthias Scheffler ◽  
Sabine Merkelbach-Bruse ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Overall Survival ◽  
Molecular Diagnostics ◽  
Clinical Data ◽  
Single Gene ◽  
Genomic Medicine ◽  
Genetic Alterations ◽  
Clinical Diagnostics ◽  
Central Platform ◽  
The Impact

e20596 Background: Using next-gen sequencing of predefined gene panels in routine clinical diagnostics of lung cancer allows, in contrast to single-gene assays, assessment of co-occuring mutations, which might underly heterogeneity of response to targeted drugs and survival. The Network Genomic Medicine (NGM) performs high sensitive next generation sequencing (NGS) based routine molecular diagnostics on a central platform for about 5000 inoperable lung cancer patients (pts) annually in Germany. Methods: NGS panel used in NGM consists of 17 genes to cover potentially targetable aberrations. Mutation analyses were run on an Illumina (MySeq) platform, while FISH analyses were performed separately. In 2016, we have started the evaluation of all NGM pts with available clinical data who had received NGS-based molecular diagnostics. In particular, we have focused on non-squamous (non-sq) and squamous (sq) NSCLC pts with co-occurring mutations: their frequency, significance and impact on overall survival. Results: From 2014 molecular genotyping was performed for 7,893 NGM pts (n = 7,246 NSCLC (5,667 non-sq and 1,487 sq pts) and n = 489 SCLC) with eligible clinical data. Genetic alterations in transformation-associated pathways were found in 79 % of all NSCLC pts. Furthermore, co-occurring mutations were detected in 39 % of these pts: 40 % in non-sq and 37 % in sq NSCLC. 11 % of pts had more than 2 co-occurring mutations. 1 % of all pts had 5 co-occurring mutations. The most frequent paired mutations were KRAS, EGFR and MET each with TP53 in non-sq and FRGF1 and TP53 in sq NSCLC. The incidences and significance of 3, 4 and 5 co-mutations as well as the impact of these co-occurring mutations on overall survival will be presented. Conclusions: Frequent occurrence of co-occuring mutations in transformation – associated pathways underlines the genetic heterogeneity also of lung cancer with classical driver mutation and the impact of co-occurring mutations on survival. This work confirms the use of molecular multiplex testing in routine molecular diagnostics of NSCLC. Assessment of co-occuring mutations will help to further specify genetically defined subgroups of lung cancer with therapeutic relevance.

Application of next generation sequencing (NGS) in gastrointestinal stromal tumor (GIST).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13675-e13675
Author(s):  
Bin Li ◽  
Jun Bai ◽  
Lingjun Zhu ◽  
Sen Zhang ◽  
Guihua Wang ◽  
...  
Keyword(s):  
Small Intestine ◽  
Pathogenic Mutation ◽  
Genetic Alterations ◽  
Resistance Mutations ◽  
Liquid Biopsies ◽  
Pik3ca Mutations ◽  
Nf1 Gene ◽  
Treatment Naïve ◽  
Exon 9 ◽  
Exon 11

e13675 Background: The majority of GISTs harbor activating mutations in KIT or PDGFRA. However, the clinical relevance of other genomic alterations, tumor mutation burden(TMB), microsatellite instability(MSI) as well as ctDNA-based clinical utility were poorly examined. Methods: A total of 106 samples obtained from 92 patients (Table) were sequenced by NGS with 1021 cancer-associated gene panel. Results: In 62 treatment naïve patients, the most common primary localization was stomach (29), followed by small intestine (22). KIT mutations were found in 52 (89.0%) cases. The most frequent alterations occurred in exon 11(48/55), followed by exon 9(6/55) and then exon 17(1/55). The primary foci of GIST patients harboring KIT exon 9 mutations was small intestine mainly. There was one patient presented with PDGFRA mutation in exon 18 (1.9%,1/62). All these cases were absent of BRAF, RAS, PIK3CA mutations, while actionable mutations detected from other genes, including CDKN2A(6/62), NF1(4/62) TSC1(2/62), PTEN(2/62) and BRCA2(2/62). Six (6/62, 10.0%) patients were KIT/ PDGFRA wild type and two of them (33.3%) detected pathogenic mutation in NF1 gene, with diagnosed age at 36 and 38 years old respectively. TMB and MSI status were analysed in 30 GIST using NGS method and no TMB-H or MSI-H sample found. Thirty-six patients who resistance to imatinib or sunitinib were screened. Nine (47.4%) and four patients (16.0%) harbored resistant mutations in KIT gene detected from tumor and blood sample cohort, concurrent sensitive and resistance mutations remain the major subtype (Table). The low percent of ctDNA detection were consistent with previous studies that clinical characteristics (tumor size, Ki67, etc.) could influence the results. Conclusions: Panel-based NGS could comprehensively elucidated the landscape of GIST and identified more tumor-specific genetic alterations which can be managed by targeted therapy. But immunotherapy and liquid biopsies remains challenging and needs further investigation from our results. [Table: see text]

scholarly journals Epidemiology of Rifampicin Resistant Tuberculosis and Common Mutations in rpoB Gene of Mycobacterium tuberculosis: A Retrospective Study from Six Districts of Punjab (India) Using Xpert MTB/RIF Assay

2016 ◽  
Vol 8 (02) ◽  
pp. 096-100 ◽  
Author(s):  
Ramandeep Kaur ◽  
Neerja Jindal ◽  
Shilpa Arora ◽  
Shajla Kataria
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Surrogate Marker ◽  
Rpob Gene ◽  
Multidrug Resistant ◽  
Resistance Pattern ◽  
Resistance Mutations ◽  
Resistant Tuberculosis ◽  
Mdr Tb ◽  
Previously Treated Patients ◽  
User Friendly

ABSTRACT Background: Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in <2 h. The RIF resistance pattern in Malwa region of Punjab, India, is not documented. Here, we report the epidemiology of RIF-resistant TB and mutations in rpoB gene of Mycobacterium tuberculosis (MTB). Materials and Methods: A total of 1612 specimens received between October 2013 and February 2015 were tested by Xpert MTB/RIF assay following manufacturer’s instructions. The results thus obtained were analyzed using SPSS version 20.0.0 (SPSS Inc., Chicago, IL, USA) statistical software. Result: RIF resistance was statistically higher in previously treated patients in comparison to the new patients (P = 0.006) and in patients with acid fast-Bacilli (AFB) positive smears to AFB-negative smears (P = 0.048). RIF resistance mutations in 130 specimens revealed frequency of E 73/130 (56%), B 28/130 (21.5%), D 18/130 (13.8%), A 11/130 (8.4%), and C 1/130 (0.7%) while in one specimen, mutation combination, i.e., mutations associated with more than one probe (A and B both) was present. Conclusion: Xpert MTB/RIF assay is a user-friendly screening tool for detection of MTB and RIF resistance from suspected TB/MDR cases in a shorter period of time. It could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of RIF resistance in MTB isolates.

HYBRIDIZATION-BASED NUCLEIC ACID AMPLIFICATION TEST TERHADAP CARTRIDGE-BASED NUCLEIC ACID AMPLIFICATION TEST TERKAIT MULTIDRUG-RESISTANT TUBERCULOSIS

2016 ◽  
Vol 21 (3) ◽  
pp. 237
Author(s):  
Ivana Agnes Sulianto ◽  
Ida Parwati ◽  
Nina Tristina ◽  
Agnes Rengga I
Keyword(s):  
Nucleic Acid ◽  
Rpob Gene ◽  
Multidrug Resistant ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Isoniazid Resistance ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis ◽  
Mdr Tb ◽  
Low Sensitivity

Indonesia has high burden of multidrug-resistant tuberculosis (MDR-TB). Cartridge-based nucleic acid amplification test (CB-NAAT), which is recommended as a diagnostic method of MDR-TB by World Health Organization, is faster in achieving the result. This method determines MDR-TB only from the rifampisin resistance, by detecting mutations that occur on the 81 bp hot-spot region of the rpoB gene. The isoniazid resistance is not included in the determination of MDR-TB by this method. Hybridization-based NAAT (HB-NAAT) detects MDR-TB not only from the rifampisin resistance (codon 526 and 531 rpoB gene), but also from the isoniazid resistance (codon 315 katG gene). The aim of this study was to know the validity of the HB-NAAT in detecting MDR-TB using sputum with CB-NAAT as the gold standard in a diagnostic study. All of 51 sputums were collected during June 2013 from patients suspected pulmonary MDR-TB at Dr. Hasan Sadikin General Hospital. The result of CB-NAAT were 16 MDR-TB, 12 TB non MDR, and 23 non TB. HB-NAAT examination results were 3 MDR-TB, 25 TB non MDR (3 RMR, 6 IMR, 16 susceptible) and 23 non TB. The sensitivity of HB-NAAT was 18.75% and specificity 100%. Low sensitivity values may due to the high mutation variations in the samples. So it could not be detected only by codons 526 and 531 for rifampisin resistance. For the detection of isoniazid resistance, HB-NAAT have optimal primer at low concentrations and it also need more than katG genes to detect isoniazid resistance. Based on this study, it can be conclued, that HBNAAT has low sensitivity but high specificity in the detecting MDR-TB.

ALK resistance mutations and co-occurring genetic alterations to the ALK tyrosine kinase inhibitors in lung cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e20675-e20675 ◽  
Author(s):  
Jin-Ji Yang ◽  
Chi Zhang ◽  
Jun Zhao ◽  
Pingping Dai ◽  
Gen Lin ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Resistance Mutation ◽  
Genetic Alterations ◽  
Driver Gene ◽  
Resistance Mutations ◽  
Significant Difference

e20675 Background: Acquired ALK mutations pose a challenge in multiple ALK tyrosine kinase inhibitors (TKIs) for lung cancer. In our study, we examined the profiles of ALK resistance mutations and co-occurring genetic alterations after targeted therapy. Methods: Using targeted gene capture and next-generation sequencing technologies, we analyzed the somatic mutations from174 patients (pts) with post-TKI samples. Among them, 123 pts received first-generation TKI crizotinib only, 51 pts (34 with second-generation TKI, 17 with third-generation TKI) treated with multiple ALK-TKIs. Results: After the treatment of ALK-TKIs, 29% (50/174) patients developed ALK resistance point mutations, including G1202R (22 pts), G1269A (13 pts), L1196M (8 pts), D1203N (5 pts), F1174L (4 pts), I1171T (4 pts), E1210K (4 pts), G1128A (3 pts), F1174C (3 pts), C1156Y (1 pts), G1123S (1 pts), I1171S (1 pts), L1152R (1 pts), and 10 of them had multi-clone. Specifically, G1269A was found a higher proportion in crizotinib group contrast to multi-TKIs cohort (10/24 vs 3/26, p = 0.024). The recalcitrant G1202R was another common resistance mutation, but there was no significant difference between the two groups (p = 0.052). Other concurrent genetic alterations related to clinical response were usually observed in TP53 mutations (46%), furthermore it seemed to be more frequently detected in post-crizotinb compared with multi-TKIs (P = 0.023). Activated bypass signaling may promote tumor progression. In non-ALK resistance point mutations samples (n = 124), co-occurring genomic alterations in EGFR (32/124, p = 0.004) were significantly more enriched in crizotinib group (n = 99). The driver gene mutation may limit crizotinib response. However, EP300 (24%), CDKN2A (12%), TRIM58 (12%), STK11 (12%) or KRAS (8%) mutations were common in the multiple ALK-TKIs group (n = 25). Conclusions: In lung cancer patients, ALK resistance point mutations G1269A was significantly enriched in post-crizotinib, while patients with multiple ALK-TKIs may frequently found G1202R or L1196M. The co-occurring genetic alterations in TP53 or EGFR after the TKIs therapeutic may offer directions for further research and therapy in lung cancer.

scholarly journals Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis

2006 ◽  
Vol 50 (8) ◽  
pp. 2640-2649 ◽  
Author(s):  
Manzour Hernando Hazbón ◽  
Michael Brimacombe ◽  
Miriam Bobadilla del Valle ◽  
Magali Cavatore ◽  
Marta Inírida Guerrero ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
Sample Selection ◽  
Multidrug Resistant ◽  
Small Sample ◽  
Inverse Association ◽  
Resistance Mutations ◽  
Isoniazid Resistance ◽  
Large Scale Analysis ◽  
Small Sample Sizes

ABSTRACT The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

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