Korean Lepr Bull.  2019 Dec;52(1):29-40. 10.33161/klb.2019.52.1.29.

Comparison of Effective DNA Extraction method for Molecular biological study of Mycobacterium leprae

• Kim YJ
• Park JH
• Kim JP

• KMID: 2466490
• DOI: http://doi.org/10.33161/klb.2019.52.1.29

Abstract

BACKGROUND
Leprosy is an important health problem in many geographical areas yet. It is caused through a cough or contact with fluid from the nose of a person infected by Mycobacterium leprae. Study of DNA from M. leprae is important to understand essentiality for leprosy. However, there is no standard in many parts, so various studies are needed. OBJECTS: In this study, DNA extraction method were confirmed for the effective detection of M. leprae. And restriction enzyme fragment length polymorphism typing and high resolution melt (HRM) analysis were performed for comparison with sequencing analysis.
METHODS
Compared with three DNA extraction methods (BB, SM and SP) with real-time polymerase chain reaction (PCR). Analysis single nucleotide polymorphism (SNP) genotype and tandem repeats by PCR amplification, and then compare with sequence.
RESULTS
BB method was effective when measuring the concentration and threshold cycle (Ct) compared with SM and SP methods. When compared with restriction fragment length polymorphism typing method and sequence analysis, all methods were suitable for SNP1 and 3 type classification. Tandem repeats values of BB method were correspond to sequence analysis than SM and SP methods in HRM analysis.
CONCLUSIONS
The DNA extraction method by bead is useful approach for studying of M. leprae.

Keyword

DNA extraction; Leprosy; Mycobacterium leprae; PCR

MeSH Terms

Classification
Cough
DNA*
Genotype
Humans
Leprosy
Methods*
Mycobacterium leprae*
Mycobacterium*
Nose
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Polymorphism, Single Nucleotide
Real-Time Polymerase Chain Reaction
Sequence Analysis
Tandem Repeat Sequences
DNA

Figure

• 

  Fig. 1 SNP subtyping of M. leprae based on PCR-RFLP9

• 

  Fig. 2 Results of DNA concentration and purity of three extraction methods for M. leprae. (A) The DNA concentration was high in BB method compared to SM and SP methods. (B) The DNA purity of BB method was high compared with SM and SP methods but was not significant.(One-way ANOVA followed by tukey-kramer's test, **p < 0.01 vs. BB method)

• 

  Fig. 3 Comparison of DNA extraction methods for M. leprae via Ct values of PCR. Low Ct value indicates high DNA amplification. PCR fluorescent dye was used as two types: (A) SYBR Green, (B) Probe. The total Ct values were significantly increased in SM and SP methods compared to the BB method. (One-way ANOVA followed by tukey-kramer's test, **p<0.01 vs. BB method)

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