[Establishment and application of quantitative detection for heteroplasmic mtDNA mutation 3243AâG].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi
; 31(4): 438-43, 2014 Aug.
Article
em Zh
| MEDLINE
| ID: mdl-25119906
ABSTRACT
OBJECTIVE:
To develop a rapid, simple, cost-effective, accurate and sensitive method for quantitative detection of mitochondrial DNA (mtDNA) 3243AâG mutation in order to provide reference for selecting the best detection method under different conditions.METHODS:
Genomic DNA was extracted from peripheral leucocytes of 17 individuals from a Wenzhou family featuring maternally inherited diabetes and deafness (MIDD). Heteroplasmic level of mtDNA 3243AâG mutation was determined respectively with polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP), real time-amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) and pyrosquencing. Eleven plasmids with various heteroplasmic levels of the 3243AâG mutation (ranging from 0 to 100%)were constructed as the standards. The reliability of above methods was compared by correlation coefficient based on observed and expected values.RESULTS:
For all three methods, measurement of the standards showed a linear correlation between the expected and detected values, i.e., PCR-RFLP (R(2)=0.828), RT-ARMS-qPCR (R(2)=0.998) and pyrosquencing (R(2)=0.997). For the MIDD family, it was consistent that there are 13 members carrying the A3243G mutation with different heteroplasmic levels. And there was no significant difference between the results by RT-ARMS-qPCR and pyrosquencing.CONCLUSION:
PCR-RFLP is not appropriate for the quantitative detection but could be used for early clinical screening. Both RT-ARMS-qPCR and pyrosquencing are suitable for the detection of low heteroplasmic level of A3243G mutation. Compared with pyrosquencing, RT-ARMS-qPCR is rapid, reliable and cost-effective, and is the best choice for detecting low mutation loads.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Tipo de estudo:
Diagnostic_studies
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Evaluation_studies
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Guideline
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Prognostic_studies
Limite:
Adolescent
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Adult
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Child
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Female
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Humans
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Male
Idioma:
Zh
Ano de publicação:
2014
Tipo de documento:
Article