Quantitative polymerase chain reaction method compared with traditional methods for Clostridium tyrobutyricum in grass-clover silage

Abstract: C. tyrobutyricum is the most common type of bacteria to contaminate milk and cause late blown cheese caused by high spore counts in silage. The purpose of this thesis was to test a qPCR method to quantify C. tyrobutyricum in silage, by investigating potential differences in the clostridia spore estimate between the qPCR method, the MPN method and the plate count method. Furthermore, relationships between silage fermentation characteristics and clostridia spore contents analysed with the different methods were evaluated. The silage that was used was from a sward containing 75% red clover and 25% grass that was harvested on the 4th of September 2011 (third harvest) at Skara in the southwest of Sweden. The silage fermentation parameters were analyzed and the C. tyrobutyricum count was analysed with Qauntitative Polymerase Chain Reaction (qPCR method), most probable number method (MPN-method) and plate count method. Relationships between clostridia spore counts from the traditional laboratory methods (the plate method and the MPN method) and the qPCR method were investigated by linear regression analysis using PROC REG in SAS Also, the least-square means of clostridia spore counts from the analytical methods were compared in the general linear model procedure of SAS (PROC GLM). Stepwise regressions on the relationships between silage fermentation characteristics and clostridia spore count from the different methods were performed. Comparisons of the least-square (LS) means of clostridia spore count from the different methods did not show any significant differences between the methods. The simple linear regressions showed that the MPN method performed at the laboratory of SLU, Skara, Sweden (MPN-S) and the plate method had moderate to strong correlations with the qPCR method, which indicates their reliability for estimating the Clostridium spore content in clover-grass silage. The MPN method performed at the LKS mbH, Germany (MPN-D) showed low correlations with the qPCR. The qPCR method showed very high correlation with the fermentation parameters compared to the MPN-D and the plate count methods. Generally, the qPCR method hade higher correlation with the fermentation parameters and smaller variations compared with MPN and plate-method. Conclusion of the study was no significant differences in Clostridia spore counts of red clover-grass silage between the qPCR, MPN-D, MPN-S and the plate methods when comparing the LS means of the Clostridia counts from the different methods. The simple linear regressions showed that the MPN-S and the plate method had moderate to strong correlations with the qPCR method. The MPN-D showed low correlations with the qPCR. The high specificity for the qPCR method decreases the risk of counting other lactate degraders than C. tyrobutyricum in the silage. The qPCR method showed very high correlation with the fermentation parameters compared to the MPN-D and the plate count methods. The MPN-S method showed promising relationships with the fermentation products in the silage.