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A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples.

ABSTRACT: Mutations of both the IDH1 and IDH2 (isocitratedehydrogenase enzyme 1 and 2) genes have recently been described in cases of human glioma. Since IDH1 mutations have been associated with better clinical outcome, they are suitable predictive markers for adult glioma patients. We have developed a pyrosequencing assay that allows both the sensitive and rapid detection of mutant IDH1 alleles in DNA extracted from formalin-fixed, paraffin-embedded tissues. PCR products that span exon 4 of IDH1 were used as a template for pyrosequencing. For validation, PCR products were additionally cloned and sequenced conventionally by Sanger sequencing. Sensitivity was measured by titration of wild-type and mutant sequences. PCR kinetic experiments were performed to investigate the influences of PCR cycle number on the accuracy of the assay. We found that a minimum of 5% of mutant IDH1 alleles can easily be detected with the pyrosequencing approach. So far, there are few data regarding IDH1 mutation status in high-grade gliomas of childhood. Therefore, we applied this assay to 47 pediatric high-grade glioma samples (age range 6 weeks to 23 years). Mutations were found in 5/14 astrocytoma III and in 6/33 glioblastomas. In conclusion, we have developed a pyrosequencing-based assay for the detection of mutations at the hotspot regions of IDH1 and provide proof for its applicability as a molecular diagnostic assay for clinical samples.

SUBMITTER: Setty P

PROVIDER: S-EPMC2963913 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples.

Setty Prashanth P Hammes Jennifer J Rothämel Thomas T Vladimirova Valentina V Kramm Christof M CM Pietsch Torsten T Waha Andreas A

The Journal of molecular diagnostics : JMD 20100916 6

Mutations of both the IDH1 and IDH2 (isocitratedehydrogenase enzyme 1 and 2) genes have recently been described in cases of human glioma. Since IDH1 mutations have been associated with better clinical outcome, they are suitable predictive markers for adult glioma patients. We have developed a pyrosequencing assay that allows both the sensitive and rapid detection of mutant IDH1 alleles in DNA extracted from formalin-fixed, paraffin-embedded tissues. PCR products that span exon 4 of IDH1 were use ...[more]

PMID: 20847279

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Project description:BACKGROUND:Yersinia pestis is a contributing agent to the epidemic disease, plague, which killed an estimated 200 million people during historical times. In this study, a rapid, cheap, sensitive, and specific technique, the lateral flow assay (F1 strips), has been successfully developed to detect this pathogen, by using paired monoclonal antibodies (MAbs) against Y. pestis capsule like fraction 1 (F1) protein. Compared with the polyclonal antibody (PAb) based F1 strips, the Mab-based F1 strips have a remarkable increased detection limitation (10 to 100 folds). Furthermore, besides the limitation and specificity evaluation, the application of this F1 strip on simulated clinical samples indicate the LFA can be a good candidate to detect plague. METHODS:Recombinant F1 antigen was expressed and purified from a series of works. The various anti-F1 monoclonal antibodies generated from hybridoma cells were screened with the ELISA technique. To evaluate the feasibility of this Y. pestis F1 test strip, the F1 protein/Y. pestis was spiked into simulated clinical samples such as human serum, mouse bronchoalveolar lavage fluids, and mouse blood to mimic natural infection status. Additionally, this technique was applied to detect the Y. pestis in the environment-captured rats, to evaluate the practical usefulness of the strips. RESULTS:By using this MAb-based-LFA technique, 4 ng/ml of recombinant F1-protein and 103 CFU/ml of Y. pestis could be detected in less than 10 mins, which is at least 10-folds than that of the PAb format. On the other hand, although various Yersinia strains were applied to the strips, only Y. pestis strain showed a positive result; all other Yersinia species did not produce a positive signal, indicating the high efficiency and specificity of the MAb-based F1-strips. CONCLUSION:Based on our findings, we suggest that the MAb-format-LFA will be valuable as a diagnostic tool for the detection of Y. pestis. This report shows that the F1 strip is sufficient to support not only the detection of plague in simulated clinical samples, but also it may be a good candidate to meet the epidemiological surveillance during an outbreak of the biological warfare.

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A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples.

Publications

A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples.

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