A One-Tube Nested Polymerase Chain Reaction for the Detection of Mycobacterium Bovis in Spiked Milk Samples: An Evaluation of Concentration and Lytic Techniques

Abstract

The objective of this study was to evaluate the use of a one-tube nested polymerase chain reaction (OTN PCR) with 5 concentration and lytic treatments for the detection of Mycobacterium bovis in experimentally inoculated milk samples (spiked samples). OTN PCR and the following treatments were tested in inoculated samples: 1) centrifugation; 2) C₁₈-carboxypropylbetaine + capture resin 1 + Proteinase K (CB18-CH-PK); 3) centrifugation + capture resin 1 + Proteinase K; 4) centrifugation + capture resin 2 + Proteinase K; and 5) centrifugation + immunomagnetic separation (IMS). The OTN PCR and the 5 treatments were evaluated in 2 different sets of spiked milk samples. One set consisted of 10-fold serial dilutions of a phenol-killed M. bovis in milk to final concentrations ranging from 5 to 50,000 cells/ml of milk. The other set of samples consisted of 2.5 serial dilutions of milk spiked with M. bovis to final concentrations ranging from 20.5 to 5,000 cells/ml of milk. Each treatment was repeated 5 times at each cell concentration. CB18-CH-PK and IMS were significantly more sensitive than other treatments. The lowest detection limit for these techniques was 20–50 cells/ml of spiked milk. The specificity of OTN PCR in this study was high as demonstrated by the lack of DNA amplification products when M. bovis cells were not present in the samples. [The OTN PCR used in conjunction with CB18-CH-PK or IMS could be effectively used as a diagnostic and/or screening test for the detection of M. bovis in milk from herds with bovine tuberculosis.]