Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer
ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniq...
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doaj-8daf050c693f4e6688e387389e22a67a2020-11-25T00:07:51ZengSociedade Brasileira de MicrobiologiaBrazilian Journal of Microbiology1678-440548474074610.1016/j.bjm.2016.11.012S1517-83822017000400740Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beerYanlin MaYang DengZhenbo XuJunyan LiuJianjun DongHua YinJunhong YuZongming ChangDongfeng WangABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822017000400740&lng=en&tlng=enPropidium monoazidePolymerase chain reactionBeer spoilage bacteriaLactobacillus brevis horA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yanlin Ma Yang Deng Zhenbo Xu Junyan Liu Jianjun Dong Hua Yin Junhong Yu Zongming Chang Dongfeng Wang |
spellingShingle |
Yanlin Ma Yang Deng Zhenbo Xu Junyan Liu Jianjun Dong Hua Yin Junhong Yu Zongming Chang Dongfeng Wang Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer Brazilian Journal of Microbiology Propidium monoazide Polymerase chain reaction Beer spoilage bacteria Lactobacillus brevis horA |
author_facet |
Yanlin Ma Yang Deng Zhenbo Xu Junyan Liu Jianjun Dong Hua Yin Junhong Yu Zongming Chang Dongfeng Wang |
author_sort |
Yanlin Ma |
title |
Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer |
title_short |
Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer |
title_full |
Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer |
title_fullStr |
Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer |
title_full_unstemmed |
Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer |
title_sort |
development of a propidium monoazide-polymerase chain reaction assay for detection of viable lactobacillus brevis in beer |
publisher |
Sociedade Brasileira de Microbiologia |
series |
Brazilian Journal of Microbiology |
issn |
1678-4405 |
description |
ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells. |
topic |
Propidium monoazide Polymerase chain reaction Beer spoilage bacteria Lactobacillus brevis horA |
url |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822017000400740&lng=en&tlng=en |
work_keys_str_mv |
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