Establishment Of Real-time PCR Methods For Detection Of Total Bacteria In Raw Milk

In recent years, the scale of milk consumption gradually increased because of milkhas a rich nutrient composition, which favours growth of a broad variety of microbialspecies. The microbes in raw milk can originate from different sources. The bacteriain raw milk can be responsible for flavour defects of dairy products or may evenconstitute a health risk. So far, research on raw milk bacteria was mainly focused onestablish a rapid method for detection of total bacteria in raw milk.In the study, direct quantifing the16Sr DNA gene from total bacteria in order toestimate target-gene copy number as a proxy of total bacteria populations byemploying the real-time PCR.500cow raw milk samples were collected from Groundlimited liability company, Jilin Provience. Application of biochemical experimentsand direct sequencing to identify the bacteria isolation from the raw milk, while acommunity spectrum was generated. The results shows that common types of bacteriacontamination of raw milk are Grignard Lactococcus, Streptococcus sanguinis,Staphylococcus intermedius, Kocuria kristinae, Staphylococcus haemolyticus,Aerococcus viridans, Enterobacter cloacae, Yokenella regensburgei, Hafnia alvei,Escherichia coli, Cronobacter sakazakii, Serratia marcescecs, Klebsiella oxytoca. Toestimating the community size which has led to the application of real-time PCRmethods using universal primers targets16S rDNA gene in order to quantify the genecopy number in the pure suspensions and the artificially contaminated samples toestablish standard curve between lgCFU and CT values. Referring to GB19301-2010,the criterion of the substandard milk was total number of bacteria in raw milk reachesto2×106CFU/g(mL).The range of CT values was obtained by analysis the CT valueswhen the concentration of the bacteria was2×106CFU/mL in the pure bacterialsuspension and the artificially contaminated samples. The results of the raw milksamples employed culture-dependent and real-time PCR approaches verified the accuracy of the real-time PCR method. The range of CT values in the pure bacteriasuspensions was27.03~30.17, while in the artificially contaminated samples was29.81~31.49. The latter range can be used as reference range in actual samples. Theaccuracy of the method was about90%and the detection time was about4h. Themethod was a new method for rapid detection of total bacteria in raw milk, and it canbe determined the quality level of the milk quickly.