Development of a real-time RT-PCR method for enumeration of viable Bifidobacterium longum cells in different morphologies

作者：Reimann Sebastian; Grattepanche Franck; Rezzonico Enea; Lacroix Christophe^*

来源：Food Microbiology, 2010, 27(2): 236-242.

DOI：10.1016/j.fm.2009.10.010

• Bifidobacterium
• qRT-PCR
• mRNA-stability
• Cell aggregation

Viability of probiotic bacteria is traditionally assessed by Plate Counting which has several limitations, including Underestimation of cells in aggregates or chains morphology We describe a quantitative PCR (qPCR)-based method for an accurate enumeration of viable cells of Bifidobacterium longum NCC2705 exhibiting different morphologies by measuring the mRNA levels of cysB and purB, two Constitutively expressed housekeeping genes Three primer-sets targetting short fragments of 57-bp of cysS and purB and one 400-bp fragment of purB were used Cell quantification of serially diluted samples showed a good correlation coefficient of R(2) 0 984 +/- 0 003 between plate counts and qRT-PCR for all tested primer sets Loss of viable cells exposed to a lethal beat stress (56 degrees C, 10. 20 and 30 min) was estimated by qRT-PCR and plate Counts No significant difference was observed using qRT-PCR targeting the 400-bp fragment of purB compared to plate Counts indicating that this fragment is a suitable marker of cell viability In contrast, the use of the 57-bp fragments led to a significant overestimation of viable cell counts (18 +/- 3 and 7 +/- 2 fold for cysB and pur-B, respectively) Decay of the mRNA fragments was studied by treatment of growing cells with rifampicin prior qRT-PCR The 400-bp fragment of purB was faster degraded than the 57-bp fragments of cysB and purB The 400-bp fragment of purB was further used to enumerate viable cells in aggregate state Cell counts were mote than 2 log(10) higher using the qRT-PCR method compared to plate Counts Growing interest in probiotic charact

• 出版日期2010-4