Genotyping with TaqMAMA

Li, Baohui; Kadura, Ibrahim; Fu, Dong-Jing; Watson, David E.

doi:10.1016/j.ygeno.2003.08.005

Cited by 83 publications

(67 citation statements)

References 13 publications

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“…One of the most important shortcomings of MAMA PCR is the fact that multiple mutations occurring in the same viral variant cannot be identified. While the detection capabilities of MAMA PCR, as well as its quantitative properties when the method is properly adapted (10,18), are unquestionable, the unfeasibility of differentiation of combinatory and single mutations taking place within the same molecule is a major drawback. Thus, usage of more advanced technology such as amplicon deep sequencing is more appropriate for a thorough evaluation of the viral population.…”

Section: Discussionmentioning

confidence: 99%

“…Using this approach, we designed MAMA PCR primers for the identification of the known drug-resistant HCV mutants. Primers bearing one single mismatch position at the penultimate position (Table 2) were designed according to the guidelines reported by others (18). Implementation of the corresponding PCR protocol was carried out using artificial constructions bearing the appropriate nucleotide mutations.…”

Section: Consensus Sequencing Does Not Reflect the Presence Of Drugrementioning

confidence: 99%

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Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

et al. 2012

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The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of selective pressure, drug-resistant hepatitis C virus (HCV) mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring drugresistant HCV mutants. MAMA PCR successfully identified the corresponding HCV variants, while conventional methods such as direct sequencing, endpoint limiting dilution (EPLD), and bacterial cloning were not sensitive enough to detect circulating drug-resistant mutants in clinical specimens. Ultradeep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment-naïve patients, the frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients, showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy.

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Section: Discussionmentioning

confidence: 99%

Section: Consensus Sequencing Does Not Reflect the Presence Of Drugrementioning

confidence: 99%

Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

et al. 2012

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“…This technique can detect 1% of a mutant allele, but additional analysis is necessary to identify the variant. Some of the most popular techniques for enriching known mutations are ARMS (amplification refractory mutation system) (15 ), peptide nucleic acid (PNA)-mediated PCR (16,17 ), locked nucleic acid (LNA)-mediated wild typeblocking (WTB) PCR (18), MAMA (mismatch amplification mutation assay) PCR (19 ), TaqMAMA (20,21 ), and use of Scorpion primers (22 ). These methods detect mutations by allele-specific PCR and note differences in quantification cycle, and can detect 0.1% of mutant alleles.…”

mentioning

confidence: 99%

Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR

Zhou¹,

Palais

Smith

et al. 2010

Clinical Chemistry

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Background: Selective amplification of minority alleles is often necessary to detect cancer mutations in clinical samples. Methods: Minor-allele enrichment and detection were performed with snapback primers in the presence of a saturating DNA dye within a closed tube. A 5′ tail of nucleotides on 1 PCR primer hybridizes to the variable locus of its extension product to produce a hairpin that selectively enriches mismatched alleles. Genotyping performed after rapid-cycle PCR by melting of the secondary structure identifies different variants by the hairpin melting temperature (Tm). Needle aspirates of thyroid tissue (n = 47) and paraffin-embedded biopsy samples (n = 44) were analyzed for BRAF (v-raf murine sarcoma viral oncogene homolog B1) variant p.V600E, and the results were compared with those for dual hybridization probe analysis. Needle aspirates of lung tumors (n = 8) were analyzed for EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] exon 19 in-frame deletions. Results: Use of 18-s cycles and momentary extension times of “0 s” with rapid-cycle PCR increased the selective amplification of mismatched alleles. A low Mg2+ concentration and a higher hairpin Tm relative to the extension temperature also improved the detection limit of mismatched alleles. The detection limit was 0.1% for BRAF p.V600E and 0.02% for EGFR exon 19 in-frame deletions. Snapback and dual hybridization probe methods for allele quantification of the thyroid samples correlated well (R2 = 0.93) with 2 more BRAF mutations (45 and 43, respectively, of 91 samples) detected after snapback enrichment. Different EGFR in-frame deletions in the lung samples produced different hairpin Tms. Conclusions: Use of snapback primers for enrichment and detection of minority alleles is simple, is inexpensive to perform, and can be completed in a closed tube in <25 min.

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“…Using the principles of the mismatch amplification mutation assay (18), primers specific to either the revertant or nonrevertant strain, amplifying each strain individually, were designed. This allows the detection of revertants and nonrevertants separately and the quantitation of PCR products as the reaction is occurring and makes it possible to test a large number of samples concurrently.…”

mentioning

confidence: 99%

Shedding and Reversion of Oral Polio Vaccine Type 3 in Mexican Vaccinees: Comparison of Mutant Analysis by PCR and Enzyme Cleavage to a Real-Time PCR Assay

et al. 2007

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A uracil-to-cytosine mutation at nucleotide position 472 of oral poliovirus vaccine type 3 (OPV3) contributes to the development of vaccine-associated paralytic poliomyelitis (VAPP). To analyze OPV3 shedding patterns, we previously used the multistep method of mutant analysis by PCR and enzyme cleavage (MAPREC). This involves conventional reverse transcription-PCR to detect OPV3, followed by a restriction digest to quantify position 472 reversion. Real-time PCR detects and quantifies nucleic acid as PCR occurs and avoids postreaction processing. The goal of this study was to compare a real-time PCR method to MAPREC. Seventy-three stool samples from Mexican OPV recipients underwent the reverse transcription-PCR step of MAPREC and real-time PCR. Real-time PCR identified 23% more OPV3-positive samples than conventional reverse transcription-PCR. When reversion was compared, the revertant proportion (RP), defined as the percentage of revertants in a sample, differed by <10% in 21/25 (84%) samples. The four samples differing by >10% were obtained within 5 days of OPV administration. The real-time PCR assay identified samples with an RP of >85% with 94% sensitivity and 86% specificity compared to MAPREC. The mean difference in RP between the two methods was 3.6% (95% confidence interval, ؊0.3 to 7.5%). Real-time PCR methods reliably detect OPV3, and reversion estimates correlate more consistently with MAPREC when OPV3 reversion rates are high. Detecting VAPP-related mutations by real-time PCR is rapid and efficient and can be useful in monitoring ongoing global polio eradication efforts.Vaccination using the trivalent, live attenuated oral poliomyelitis vaccine (OPV) led to the elimination of poliomyelitis in the United States and several regions of the world (15, 26). Specifically, OPV is transmitted by the fecal-oral route and by contact with pharyngeal secretions and thereby confers immunity to unvaccinated individuals (10). However, OPV strains also undergo mutations and recombination during replication in humans (13), with the attenuating OPV sequences often being selected against in the human intestinal tract (21). The transmission of these revertant strains leads to vaccine-associated paralytic poliomyelitis (VAPP) (2).Although many industrialized countries have adopted inactivated polio vaccine (IPV) into their immunization programs due to the risk of VAPP (7), several resource-poor nations cannot afford this policy change. Currently, the global VAPP burden is estimated to be 250 to 500 cases annually (14), with most cases occurring in resource-poor settings, and the risk of paralytic polio due to OPV will continue until as long as the oral vaccine is in use.Most cases of VAPP in the United States and in Latin America were due to serotype 3 (1, 7). A point mutation at position 472 of Sabin OPV3 within the 5Ј noncoding region and its internal ribosomal entry site is known to be an attenuating sequence for this serotype, and reversion of this nucleotide has been associated with VAPP (9). These back-mutations of ...

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Genotyping with TaqMAMA

Cited by 83 publications

References 13 publications

Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR

Shedding and Reversion of Oral Polio Vaccine Type 3 in Mexican Vaccinees: Comparison of Mutant Analysis by PCR and Enzyme Cleavage to a Real-Time PCR Assay

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