Figure 1.
Adaptation of immuno-PCR for the detection of Y. pestis antigen in dental pulp extracted from ancient teeth.
Figure 2.
Determination of the detection limits of ELISA and iPCR using various dilutions of the Y. pestis antigen.
Because the Ct values obtained by iPCR were inversely proportional to the antigen concentrations, delta-Ct values were calculated by subtracting the Ct values from the maximum cycle number of 40. Mean value and standard deviation of 2 different experiments are represented.
Figure 3.
A: Blind detection of the Y. pestis antigen in 46 coded teeth, including 34 historically positive teeth and 10 negative teeth, using ELISA (left) and iPCR (right). For each tooth, the mean value of 2 independent experiments was represented. Dotted line are the cut-off for ELISA (OD = 0.196.) and for iPCR (ΔCt = 16.13). Median, 25% and 75% percentiles are represented for each group and values are the following: by ELISA, negative teeth (0.143, 0.102, and 0.159), historically positive teeth (0.146, 0.130, and 0.174), historically positive teeth and positive by PCR (0.145, 0.115, and 0.158). By iPCR: negative teeth (13.01, 12, and 14.66), historically positive teeth (15.59, 14.29, and 19.53) and historically positive teeth and positive by PCR (0.145, 0.115, and 0.158). B: Comparison of iPCR, ELISA and PCR sensitivities for the detection of the Y. pestis antigen in ancient teeth.
Figure 4.
Venn diagram representing the number of positive teeth detected by PCR, iPCR and ELISA.
Figure 5.
Molecular (losenge) and immunological (bubbles) detection of the plague agent Yersinia pestis in ancient burial sites in Europe in bones (star) and dental pulp (without star).
For immunological detection, pink bubbles represent Rapid Diagnostic Test (RDT) detection, white bubbles represent iPCR detection, green bubbles represent immunohistochemical (IH) detection and purple bubbles represent ELISA detection. 27 burial sites are represented and references are between brackets: 1. Marseille [15], [23], [31], [41]; 2. Martigues [23], [41]; 3. Etang de Berre [31]; 4. La Chaize- le-Vicomte [42]; 5.Poitiers ([42]; 6. Draguignan [23], [31]; 7. Saint-Maurice [43]; 8.Briançon [24]; 9.Lariey ([31], present study); 10. Lambesc [15], [23], [31]; 11. Vilarnau [44]; 12. Bondy ([32], present study); 13. Montpellier [16], [33]; 14. Dreux [33]); 15.Vienne [41]; 16. Sens ([33], present study); 17.Saint-Laurent [19]; 18. Venise ([19], present study); 19.Genoa [24]; 20.Parme [19]; 21.Stuttgart [34]; 22. Aschheim [17]; 23.Manching-Pichl [45]; 24.Augsburg [19]; 25. Bergen-op-Zoom [19]; 26. Hereford [19]; 27.Bourges (present study).
Figure 6.
Strategy to detect the Y. pestis antigen in ancient specimens by ELISA and iPCR.
In total, 34 ancient teeth were collected from skeletons that were excavated at five archaeological sites covering a broad time span from the 5th to 16th centuries. Dental pulp was extracted from 34 historically positive teeth and 20 negative teeth, which included modern and ancient teeth. The cut-off values for ELISA and iPCR were determined based on the screening of 10 negative teeth. Finally blind screening of 46 coded teeth, including the 34 historically positive teeth, 10 negative teeth and 2 blank controls was performed.