Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death

Abstract

Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague.

Figure 1

Photography (a), radiography (b), and recovery (c) of ancient dental pulp from the skeleton of a 1348-Black Death child confirmed as having died from the Y. pestis plague.

Figure 2

Agarose gel stained with ethidium bromide showing the 147-bp amplified Y. pestis pla fragment obtained from ancient DNA after suicide PCR. Products resulting from DNA extracted from the dental pulp tissues collected from the ancient child skeletons (lanes 3, 5, 8, and 10), negative control dental pulp tissues (lanes 4, 9, 11, and 12), and mock extraction controls (lanes 2, 6, and 7) are shown. Lanes 1 and 13 are molecular weight marker fragments. The measured molecular weight of the amplicon is indicated in the margin.

Figure 3

Agarose gel stained with ethidium bromide showing the 148-bp amplified Y. pestis pla fragment obtained from ancient DNA after suicide PCR. Products resulting from DNA extracted from the dental pulp tissues collected from the ancient adult male skeleton (lanes 2 and 3), the ancient adult female skeleton (lanes 5 and 6) and negative control dental pulp tissues (lane 8) are shown. Lanes 1, 4, 7, and 9 are molecular weight marker fragments. The molecular weight of the marker is indicated in the margin.

Figure 4

Partial sequence of the Y. pestis pla gene determined in two adult Medieval Black Death victims. The mutation identified in codon 178 and the resulting amino acid substitution are in bold.