Matrixlysis, an improved sample preparation method for recovery of Mycobacteria from animal tissue material

Abstract

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, is the main causative agent of bovine tuberculosis in alpine regions. Bacterial culture is the gold standard in bovine tuberculosis diagnostic but takes up to twelve weeks. This increases the time and costs for stocks affected with bovine tuberculosis. Hence this study focused on the implementation of a fast and precise mycobacterial detection method and compared it with currently used methods. Matrix lysis is a chemical lysis using high concentrations of urea to solubilize bovine and red deer tissue and was used to detect even smallest amounts or non-visible lesions of mycobacteria. A total of 64 samples collected from 44 animals (37 red deer and 7 cattle) were tested by Matrix lysis. Forty-three of these samples were used for Mycobacterium tuberculosis complex detection by quantitative PCR and other 21 for subtyping the genetically different variants of M. caprae. Furthermore, three Matrix lysis samples were used for Next Generation Sequencing. Our results confirm that Matrix lysis is a fast and precise method for detecting Mycobacterium tuberculosis complex in native tissue samples. However, at the moment it reaches its limits when the samples were analyzed by Next Generation Sequencing and RD4 subtyping.

Fig 1. Workflow of samples.

Fig 2. Statistical analysis showing ct-values and standard deviations of sample sets 2, 5, 10, 16B, 22B and 25C.

ML of sample 25C was performed twice using 2 gram and 4 gram tissue material, respectively.

Fig 3

Average ct-values and deviations of pathoscore groups 1 & 2 (A) and 3 & 4 (B).

Fig 4

RD4 deletions (A) and classical PCR patterns (B) of alpine M. caprae subtypes Allgäu. Lechtal and Karwendel.

Fig 5

Identification of RD4 subtype Lechtal of red deer samples 32, 33, 34 and 35 derived from ML analysis (A) and bacterial isolates (B).