Central European Journal of Medicine

, Volume 9, Issue 6, pp 839–848 | Cite as

Sensitive quantification of mitochondrial mutation using new Taqman probes

  • Hue Thi Truong
  • Van-Anh Thi Nguyen
  • Hong-Loan Thi Nguyen
  • Van-Anh Pham
  • Tuan-Nghia Phan
Research Article


The A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R2 > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R2 of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria.


A3243G mutation Mitochondrial DNA MELAS syndrome Real-time PCR Locked nucleic acid probe 


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  1. [1]
    Masakazu M., Hideyuki H., Takashi I., Hiroshi I., Susumu F., Manami A., et al., Different effects of novel mtDNA G3242A and G3244A base changes adjacent to a common A3243G mutation in patients with mitochondrial disorders. Mitochondrion, 2009, 9, 115–122CrossRefGoogle Scholar
  2. [2]
    Bai R.K., Wong L.J.C., Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach. Clin. Chem., 2004, 50, 996–1001PubMedCrossRefGoogle Scholar
  3. [3]
    Ma Y., Fang F., Yang Y., Zou L., Zhang Y., Wang S., et al., The study of mitochondrial A3243G mutation in different samples. Mitochondrion, 2009, 9, 139–143PubMedCrossRefGoogle Scholar
  4. [4]
    Smith M.L., Hua X.Y., Marsden D.L., Liu D., Kennaway N.G., et al., Diabetes and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS): radiolabeled polymerase chain reaction is necessary for accurate detection of low percentages of mutation. J. Clin. Endocrinol. Metab., 1997, 82, 2826–2831PubMedGoogle Scholar
  5. [5]
    Tsukuda K., Suzuki Y., Kameoka K., Osawa N., Goto Y.I., Katagiri H., et al., Screening of patients with maternally transmitted diabetes for mitochondrial gene mutations in the tRNA Leu(UUR) region. Diabet. Med., 1997, 14, 1032–1037PubMedCrossRefGoogle Scholar
  6. [6]
    White H.E., Durston V.J., Seller A., Fratter C., Harvey J.F., Cross N., Accurate detection and quantitation of heteroplasmic mitochondrial point mutation by pyrosequencing. Genet. Test., 2005, 9, 190–199PubMedCrossRefGoogle Scholar
  7. [7]
    Urata M., Wakiyama M., Iwase M., Yoneda M., Kinoshita S., Hamasaki N., et al., New sensitive method for the detection of the A3243G mutation of human mitochondrial deoxyribonucleic acid in diabetes mellitus patients by ligation-mediated polymerase chain reaction. Clin. Chem., 1998, 44, 2088–2093PubMedGoogle Scholar
  8. [8]
    Tajima H., Sueoka K., Moon S.Y., Nakabayashi A., Sakurai T., Murakoshi Y., et al., The development of novel quantification assay for mitochondrial DNA heteroplasmy aimed at preimplantation genetic diagnosis. J. Assist. Reprod. Genet., 2007, 24, 227–232PubMedCentralPubMedCrossRefGoogle Scholar
  9. [9]
    Singh R., Ellard S., Hattersley A., Harries L.W., Rapid and sensitive real-time polymerase chain reaction method for detection and quantification of 3243A-G mitochondrial point mutation. J. Mol. Diagn., 2006, 8, 225–230PubMedCentralPubMedCrossRefGoogle Scholar
  10. [10]
    Souza A.C., Ferreira R.C., Gonçalves S.S., Quindós G., Eraso E., Bizerra F.C., et al., Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR. J. Clin. Microbiol. 2012, 50, 2310–2314PubMedCentralPubMedCrossRefGoogle Scholar
  11. [11]
    Li Q., Yuan Y.Y., Huang D.L., Han D.Y., Dai P., Rapid screening for the mitochondrial DNA C1494T mutation in a deaf population in China using realtime quantitative PCR. Acta Otolaryngol., 2012, 132, 814–818PubMedGoogle Scholar
  12. [12]
    Wang J.Y., Gu Y.S., Wang J., Tong Y., Wang Y., Shao J.B., et al., MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR. J. Zhejiang Univ. Sci. B, 2008, 9, 610–615PubMedCentralPubMedCrossRefGoogle Scholar
  13. [13]
    Trinh L.P., Chu V.M., Phan T.N., Detection of point mutation A3243G of MELAS syndrome using modified PCR-RFLP. J. Genet. Appl. 2009, 4, 6–9, (in Vietnamese)Google Scholar
  14. [14]
    Letertre C., Perelle S., Dilasser F., Arar K., Fach P., Evaluation of the performance of LNA and MGB probes in 5’ nuclease PCR assays. Mol. Cell Probes 2003, 17, 307–311PubMedCrossRefGoogle Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hue Thi Truong
    • 1
  • Van-Anh Thi Nguyen
    • 1
  • Hong-Loan Thi Nguyen
    • 1
  • Van-Anh Pham
    • 2
  • Tuan-Nghia Phan
    • 1
  1. 1.Key Laboratory of Enzyme and Protein TechnologyVNU University of ScienceThanhxuan, HanoiVietnam
  2. 2.Department of NeurologyNational Hospital for PediatricsDongda, HanoiVietnam

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