Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis

Abstract

The aim of the study was to develop a sensitive method using quantitative real-time polymerase chain reaction (qPCR) with pooled fecal samples for the screening of Johne's disease (JD). Manufacturer-specified and our new pooling method in combination with five commercial kits for DNA extraction and purification were compared. Different volumes of pooled fecal suspensions were tested, and the results were compared for individual samples and three pool sizes (5, 10, and 50 samples); each of the fecal suspensions, which were prepared from healthy dairy and beef cattle was spiked with 0, 10, 100, or 1000 cultured Mycobacterium avium subspecies paratuberculosis (MAP) organisms or was mixed with fecal suspensions from experimentally infected cattle. The MAP DNA detection proportion with our pooling method in combination with Johne-Spin kit (Fasmac, Japan) was 100% for all models and all pool sizes, except for the low shedder model with a pool size of 50. There was no loss of sensitivity in pools of 10 subjects or less by using the new method. These results suggest that new method is a sensitive, practical, and cost-effective screening test for the detection of MAP-infected cattle and the monitoring of JD-free herds.

Keywords: Commercial DNA extraction kits; IS900; Mycobacterium avium subspecies paratuberculosis; pooled fecal samples; real-time PCR; screening test.

Figure 1

The kit A manufacturer‐specified and our fecal pooling protocol. The kit A protocol as general pooling protocol is that a fixed sample volume is used for DNA extraction, therefore which means those the Mycobacterium avium subspecies paratuberculosis (MAP) concentration in the fecal samples decreased and PCR inhibitors concentration in the fecal samples is as same as individual samples by the pooling process. Our fecal pooling protocol is the higher sample volume used for the DNA extraction and the resultant increased pooled sample volume, therefore which means those PCR inhibitors in the fecal samples increased and the MAP concentration in the fecal samples is as same as individual samples by the pooling process.

Figure 2

Comparison of the Mycobacterium avium subspecies paratuberculosis (MAP) DNA proportions of four commercial DNA extraction kits (kits A–D) for fecal samples spiked with cultured MAP. The final concentration of MAP organisms per tube was 10(A), 100(B), and 1000(C). DNA extraction kit was kit A (□), kit B (■), kit C (), kit D (). For all kits, IS900 PCR analysis was used. Values in groups marked “a” were significantly different from those in groups marked “b” (P < 0.01), and from those in groups marked “c” (P < 0.05), but values in groups marked “b” were not significantly different from those in groups marked “c” (P > 0.05). The number of samples per group was three expect the group of 1000 MAP organisms, kit B, and pool sizes of 50. The number of those groups was two because one sample was omitted due to a defective spin column.