Quantification of total viable bacteria on fish fillets by using ethidium bromide monoazide real-time polymerase chain reaction

被引:35
作者
Lee, Jung-Lim [1 ]
Levin, Robert E. [1 ]
机构
[1] Univ Massachusetts, Dept Food Sci, Massachusetts Agr Expt Stn, Amherst, MA 01003 USA
关键词
EMA; ethidium bromide monoazide; real-time PCR; quantification; fish fillet; viable and dead bacteria;
D O I
10.1016/j.ijfoodmicro.2007.07.048
中图分类号
TS2 [食品工业];
学科分类号
0832 ;
摘要
Real-time PCR based on universal primers for amplification of a highly conserved bacterial 16S rDNA sequence was utilized in conjunction with the treatment of extracted bacterial cells with ethidium bromide monoazide (EMA) for the differential enumeration of viable and dead cells on cod fillets. Amplification of DNA from dead bacterial cells was successfully inhibited by EMA, whereas the DNA from viable cells was readily amplified. The detection range of the EMA real-time PCR assay was linear from 1 x 10(1) to 1 x 10(5) mixed bacterial genomic targets per PCR derived from broth cultures of fish tissue. The minimum detection limit of bacteria was found to be 1 x 10(1) genomic units/real-time PCR, equivalent to I x 105 CFU per gram of tissue. The EMA real-time PCR allowed construction of a standard curve obtained by plotting the log of genomic targets from strictly viable cells against resulting PCR cycles (Ct values) that facilitated quantification of total viable bacteria from fish fillets. The log of the total number of genomic DNA targets from EMA treated cells and plate counts from six randomly procured cod fillets were found not to be statistically different with the exception of one fillet. The process of freezing and thawing fillet tissue resulted in a drop in mean colony forming units (CFU) detected by plate counts from log 8.5 +/- 0.2 to log 8.1 +/- 0. 1. A similar reduction in genomic targets from 8.5 +/- 0.1 to 8.0 +/- 0.16 was detected by EMA real-time PCR. (C) 2007 Elsevier B.V. All rights reserved.
引用
收藏
页码:312 / 317
页数:6
相关论文
共 24 条
[1]  
Abolmaaty A, 2000, MICROBIOS, V101, P181
[2]  
BEAUCHAT LR, 1995, J FOOD PROTECT, V59, P204
[3]   METABOLIC AND STRUCTURAL SITES OF DAMAGE IN HEAT-INJURED AND SANITIZER-INJURED POPULATIONS OF LISTERIA-MONOCYTOGENES [J].
BUNDUKI, MMC ;
FLANDERS, KJ ;
DONNELLY, CW .
JOURNAL OF FOOD PROTECTION, 1995, 58 (04) :410-415
[4]   DETECTION AND INCIDENCE OF SPECIFIC SPECIES OF SPOILAGE BACTERIA ON FISH .2. RELATIVE INCIDECE OF PSEUDOMONAS PUTREFACIENS AND FLUORESCENT PSEUDOMONADS ON HADDOCK FILLETS [J].
CHAI, T ;
CHEN, C ;
ROSEN, A ;
LEVIN, RE .
APPLIED MICROBIOLOGY, 1968, 16 (11) :1738-&
[5]   Viability of the Nonculturable Vibrio cholerae O1 and O139 [J].
Chaiyanan, S ;
Chaiyanan, S ;
Huq, A ;
Maugel, T ;
Colwell, RR .
SYSTEMATIC AND APPLIED MICROBIOLOGY, 2001, 24 (03) :331-341
[6]   Contamination and sensitivity issues with a real-time universal 16S rRNA PCR [J].
Corless, CE ;
Guiver, M ;
Borrow, R ;
Edwards-Jones, V ;
Kaczmarski, EB ;
Fox, AJ .
JOURNAL OF CLINICAL MICROBIOLOGY, 2000, 38 (05) :1747-1752
[7]   QUALITATIVE AND QUANTITATIVE CHARACTERIZATION OF SPOILAGE BACTERIA FROM PACKED FISH [J].
DALGAARD, P .
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 1995, 26 (03) :319-333
[8]   Application of real-time PCR and RT-PCR assays for the detection and quantitation of VT 1 and VT 2 toxin genes in E-coli O157:H7 [J].
Fitzmaurice, J ;
Glennon, M ;
Duffy, G ;
Sheridan, JJ ;
Carroll, C ;
Maher, M .
MOLECULAR AND CELLULAR PROBES, 2004, 18 (02) :123-132
[9]   Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7 [J].
Fortin, NY ;
Mulchandani, A ;
Chen, W .
ANALYTICAL BIOCHEMISTRY, 2001, 289 (02) :281-288
[10]   Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA [J].
González, I ;
García, T ;
Fernández, A ;
Sanz, B ;
Hernández, PE ;
Martín, R .
JOURNAL OF APPLIED MICROBIOLOGY, 1999, 86 (02) :231-236