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Yun-Ji Kim
, Jin-Ho Park, Jong-Pill Kim
, Jin-Ho Park, Jong-Pill Kim
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.
Figures and Tables
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)
Table 6
Comparison with restriction fragment length polymorphism typing method and sequence analysis for SNP types. (A) Results of restriction fragment length polymorphism typing method by sequence analysis, (B) Results for rates of TPR and TNR. All methods were significantly correspond compared to the sequence analysis (Two way followed by Likelihood Ratio Fisher's Exact, †α<0.05 vs sequence analysis). TPR and TNR values of SNP1 and 3 type were 1.00 in BB, SM and SP methods except TPR value of SNP1 type was 0.75 in SM method (Single Binary Diagnostic Test. ‡TPR value>0.80, and §TNR value>0.80 vs. sequence analysis).
Table 7
Comparison of HRM and sequence analysis for tandem repeats. (A) Results of HRM analysis by sequence analysis, (B) Results for rate of TPR and TNR. All methods were significantly independent compared to the sequence analysis (Two way followed by Likelihood Ratio Fisher's Exact). TPR and TNR values of SNP1 type and 3 type were below in general (Single Binary Diagnostic Test. ‡TPR value>0.80, and §TNR value>0.80 vs. sequence analysis).
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