PCR and DGGE Detection Limits for Wine Spoilage Microbes

Abstract

In this study the culture-independent technique, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE), was investigated for the early detection and identification of possible spoilage microbes in wine. PCR and DGGE conditions were successfully optimised with the universal primers HDA1 GC and HDA2, the bacteria-specific primers WBAC1 GC -WBAC2, and the yeast-specific primers NL1 GC and LS2. PCR and DGGE detection limits were determined for Lactobacillus plantarum, Pediococcus pentosaceus, Acetobacter pasteurianus and Brettanomyces bruxellensis when inoculated into sterile saline solution (SSS) and white wine at 10 6 cfu/mL respectively. PCR detection limits were more sensitive (10 1 to 10 2 cfu/mL) than DGGE detection limits (10 1 to 10 4 cfu/ mL), with the exception of B. bruxellensis, which had higher PCR and DGGE detection limits than the other reference microbes. PCR-DGGE analysis was also used successfully to detect and identify Lb. plantarum, A. pasteurianus and B. bruxellensis at a concentration of 10 8 cfu/mL as part of mixed populations in SSS and white wine. PCR detection limits of 10 1 cfu/mL were determined for all three reference microbes in mixed populations. The DGGE detection limits were higher for mixed populations when compared to single strains.

Impact of Chemical and Biological Fungicides Applied to Grapevine on Grape Biofilm, Must, and Wine Microbial Diversity

“…Moreover, the two methods of analysis do not generally give the same results for the same sample. The detection limits of PCR-DGGE technique have been usually considered higher than culture-dependent techniques even more with mixed populations ( Bester et al, 2010 ). Nevertheless, in previous studies it was determined that, even with counts lower than 10 1 CFU/mL, this culture-independent method provided interesting results ( González-Arenzana et al, 2017b ).…”

Intestinal microbiome changes of Ulomoides dermestoides (Chevrolat, 1878) fed with a film based on thermoplastic cassava starch and polylactic acid

“…DNA from one gut per sample was amplified using the following pair of specific primers WBACC1 GC (5’‐CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCC CCC GGT CGT CAG CTC GTG TCG TGA GA‐3’) and WBAC2 (5’‐CCC GGG AAC GTA TTC ACC GCG‐3’) (Bester et al., 2010). The final PCR reaction volume was 50 uL, containing: 29 µL of sterile deionized water, 7 µl of 10X/MgCl2 50 mmol –1 buffer, 1 µl of dNTP's 10 mmol –1 , 4 µl primers WBAC1CG and WBAC2 10 mmol –1 , 1 µl of Taq polymerase paq‐5000 (Aligent Technologies) and 4 µl of DNA 100 ng ul –1 .…”