DNA Shielding And QPCR Based Live Bacteria Enumeration Of Lactobacillus Strains At The Level

Since the functional properties and growth characteristics of probiotic bacteria are strainspecific,enumeration of a specific strain of the same species is crucial and an existing challenge.In this paper,strain specific primers were designed of 2 Lactobacillus rhamnosus by gene family analysis,and a selective counting method at strain level was established using qPCR technology to provide technical support for dynamic monitoring of the activity change pattern of strains in mixed bacterial fermentation system.The specific research contents are as follows:(1)Design and validation of primers for strain identification.In this paper,whole genome sequencing was performed for two strains of Lactobacillus rhamnosus,and gene family analysis was performed with L.rhamnosus GG and L.rhamnosus Lc705 as reference strains.Thirty-one and 19 strain-specific open reading frames(ORFs)were identified in L.rhamnosus hsryfm 1301 and L.rhamnosus LV108,respectively.Multiple primer pairs were designed within the strain-specific ORFs(and their adj acent regions),and it was verified that the strainspecific primer pairs were able to rapidly distinguish L.rhamnosus LV108(primer pairs V2,V4,V5 and V9)or L.rhamnosus hsryfm 1301(primer pairs H1,H2,H3 and H4)from the other 12 L.rhamnosus strains.(2)Design of primers for strain enumeration.In this paper,three pairs of primers(L1,L2,L3 and Y1,Y2,Y3)were designed for L.rhamnosus LV108 and L.rhamnosus hsryfm 1301,respectively,within the amplification zone of the strain identification primers.L2 and Y3 were selected according to the amplified bands for standard curve plotting.By constructing the standard quality plasmids pMD-L2 and pMD-Y3,the standard curve equation of L.rhamnosus hsryfm 1301 was obtained as y=-3.3357x+44.983(R2=99.88%,e=0.986)and that of L.rhamnosus LV108 as y=-3.411x+45.635(R2=99.95%,e=0.964).L2 and Y3 had no amplification response to Lactobacillus helveticus,Lactobacillus plantarum,Lactobacillus fermentans,Lactobacillus acidophilus,Lactobacillus case,and interfering strains such as L.rhamnosus GG and HN001,suggesting that they had good strain specificity.(3)Strain level enumeration of L.rhamnosus.In this paper,we optimized the DNA extraction process during strain counting:cells were collected from 500 μL of bacterial suspension;Take 500 μ L bacteria suspension,collect bacteria,and extract DNA by using bacterial genome DNA rapid extraction kit,treated in 180 μ of lysozyme(20 mg/mL)for 2 hours,continue to use of proteinase K(20 mg/mL)for 30 min,followed by the instructions.Enumeration results obtained by qPCR method were consistent with OD600 values when single bacteria were cultured.Since the qPCR method detected both dead and non-culturable organisms,the count results in the later growth phase were greater than those by the plate count method.In the mixed culture,qPCR successfully counted the strains in the mixed system and found antagonism between the strains.The differences between the results obtained by PMA-qPCR method and plate counting method were decreased.However,the results of PMAqPCR method were still higher than plate counting at the decay stage.In conclusion,this paper established a method to design primers for strain-specific identification,which realized the selective counting of different strains at strain level and made it possible to monitor the strains in a mixed culture system with multiple strains.