Cultivation-free sample preparation and DNA purification for direct real-time qPCR of intracellular or spore-like Coxiella burnetii in beef, goat, and lamb meat

Abstract

Coxiella burnetii is a zoonotic pathogen that has been associated with foodborne outbreaks in products with ruminant origins. However, a method to detect C. burnetii in meat has been merely studied, and commercial kits cannot efficiently fulfill this purpose. In this study, an in-house preparation method for direct real-time qPCR of C. burnetii in beef, goat, and lamb meat was designed. In the sample preparation step (step 1), trypsin digestion and cell disruption techniques were introduced to target C. burnetii in an obligate intracellular or spore-like form. Afterward, 16 DNA purification protocols involving the following steps (steps 2-3) were assessed: the precipitation of meat proteins (step 2; using 2.5, 5.0 M NaCl or 1:1, 2:1 ethanol as the precipitant) and binding of DNA to silicon dioxide particles with chaotropic salts (step 3; using 2.5, 5.0 M NaCl or 2.5, 5.0 M guanidine thiocyanate as the salt). The protocols with superior performance in high-spiked loins (estimated 4-5 log cells/g) were verified in low-spiked (1-2 log cells/g) or Bacillus thuringiensis spore-inoculated (1-2 log CFU/g) loins, ribs, and hind legs. During the protein precipitation, 5.0 M NaCl induced significantly lower protein level as demonstrated by A₂₈₀, when compared to 2.5 M NaCl or ethanol (P < 0.05). For the DNA binding step, Ct values were lowered in high-spiked goat or lamb loins (3.5-6.0▾; P < 0.05) when the concentration of NaCl was doubled or guanidine thiocyanate was introduced instead of NaCl as a chaotropic salt. Based on these results, two protocols using 5.0 M NaCl as the protein precipitant and 5.0 M NaCl (N2 + N2) or guanidine thiocyanate (N2 + G2) as the chaotropic salt were selected, which demonstrated successful detection in low-spiked (Ct values of N2 + N2, 32.9-35.6; N2 + G2, 32.3-36.4) or spore-inoculated meat (N2 + N2, 30.9-37.5; N2 + G2, 29.7-32.7). Verification in low-spiked meat showed that meat type/part significantly impacted the Ct values of N2 + G2 but not those of N2 + N2. To our knowledge, this is the first study that developed a highly accessible method for detecting C. burnetii in meat which could reveal the possibility of meat-borne Q fever in humans.

Keywords: Coxiella burnetii; DNA purification; Matrix-targeted detection; Q fever; Ruminant meat; Sample preparation.