Detection of Mycobacterium leprae DNA from Archaeological Skeletal Remains in Japan Using Whole Genome Amplification and Polymerase Chain Reaction

BACKGROUND: Identification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. However, it is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate Mycobacterium leprae DNA from archaeological skeletal remains in Japan by polymerase chain reaction, DNA sequencing and single nucleotide polymorphism (SNP) analysis. In addition, we have established a highly sensitive method of detecting DNA using a combination of whole genome amplification and polymerase chain reaction, or WGA-PCR, which provides superior sensitivity and specificity in detecting DNA from trace amounts of skeletal materials. CONCLUSION/SIGNIFICANCE: We have detected M. leprae DNA in archaeological skeletal remains for the first time in the Far East. Its SNP genotype corresponded to type 1; the first detected case worldwide of ancient M. leprae DNA. We also developed a highly sensitive method to detect ancient DNA by utilizing whole genome amplification

Geographical representation of excavation site.

<p>Location of <i>Hatanai</i> site in northeastern Japan (A). A map of seven cemeteries and the site where SK26 was excavated (B). A grave pit of SK26 at excavated (C) Cemetery 5 (not shown in this map) locates 50 m south of cemetery 2. All these figures were modified with permission from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone.0012422-Takigawa1" target="_blank">[23]</a>.</p

PCR detection of <i>M. leprae</i> DNA from skeletal samples.

<p>PCR analysis was performed using <i>M. leprae</i>-specific <i>hsp-70</i> and <i>16S-rRNA</i> primers for the DNA samples listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone-0012422-t001" target="_blank">Table 1</a>. PCR products were evaluated by 2% agarose gel electrophoresis. Human <i>β-globin</i> gene was also PCR amplified as a control for DNA preparation from a premolar root. NC1: a negative control for DNA purification in which DNase/RNase-free water was used as a sample for DNA extraction; NC2: a negative control for PCR in which DNase/RNase-free water was used instead of a DNA sample for PCR reaction; and PC: positive control DNA from <i>Thai 53</i> strain of <i>M. leprae</i>.</p