260 Modern Food Microbiology
LAMP—loop-mediated isothermal amplification
Rep-PCR—surveys repetitive sequences only
ERIC—enterobacterial repetitive intergenic concensus
Molecular Beacon—uses fluorescently labeled oligonucleotides in a qPCR format
QC-PCR—quantitative competitive
VNTR—variable number of tandem repeats (minisatellites)
Real-time PCR (qPCR, RTi-PCR)—PCR results in real time. TaqMan is a fluorogenic 5
nuclease
assay that produces results in real time.
A few applications of some of the above are summarized below.
FISH (fluorescent in situ hybridization) is an RNA probe method that targets 23S rRNA. To detect
Salmonella spp. in foods, two established oligonucleotide probes (Sal-1 and Sal-3) and a newly
constructed one were used. The probes were labeled with Cy3 (indocarboxyanine) and applied to
cultures of whole cells on slides.
55 Fluorescence occurred when the probes hybridized with the 23S
rRNA of target organisms (16S RNA sequences are more often used for bacteria). After slides were
prepared, they were counter-stained with DAPI ([4
, 6
-diamidino-2-phenylindole; a non-intercalating,
DNA-specific stain that fluoresces blue or bluish-white when bound to DNA and excited with light of
wavelength 365 nm),
109 and fluorescence was detected by epifluorescence microscopy. When FISH
was compared to a culture method for detecting Salmonella spp. in 18 different foods, up to 56
were found positive by at least one of the three FISH probes but only 30 of the 225 samples were
found positive by the culture method.
55 Overall, FISH detected salmonellae in 64% of the naturally
contaminated samples while culture methods detected in 13%. Of the 52 tested Salmonella serovars,
FISH hybridized with all 52. FISH was negative when tested on 46 non-salmonellae strains of 22
Enterobacteriaceae species, and on 14 species of 12 non-Enterobacteriaceae. Living, dead, and VBNC
(viable but nonculturable cells) could be detected. Among viable cells, those exposed to unfavorable
environmental conditions were detectable even though they may have been nonculturable. In this
study, investigators could detect salmonellae in food specimens 2–3 days sooner than by culturing.
Although it is unclear as to the minimum number of cells needed for a positive FISH response, this
number is probably >10
4 cfu/ml since it is a microscopic method.
LAMP is an in situ DNA amplification technique for the microscopic detection of bacteria. It is
similar to FISH in that they are microscope slide methods that emit fluorescence from Cy3- or DAPIstained cells. LAMP employs a low molecular weight DNA polymerase that enters whole cells; a
reaction temperature of 63
◦ C that makes it possible to use fluorescent antibody (FA) for simultaneous
cell detection; and generates large tandem repeats that prevent amplicons from leaking out of cells.
134
After DNA amplification using primers for the E. coli 0157:H7 stx 2 gene, the identification of these
cells was possible with FA labeling.
qPCR is a method whereby amplified gene products are detected by using fluorescence probes
during PCR cycling, and results can be obtained in 30–90 minutes. A number of fluorescent probes are
used such as SYBR Green I and fluorescence resonance energy transfer (FRET). These probes allow
the monitoring of the complete process, and they obviate the need for electrophoresis of end products.
FRET is a short oligonucleotide that is complementary to one DNA strand.
215 When this method was
employed to detect Giardia lamblia (the β-giardin gene was targeted) and Cryptosporidium parvum
(COWP gene was targeted), one cyst of the former and 100 cysts of the latter could be detected in
water and sewage samples.
74
Using SYBR Green I, melting curve analyses were made at the end of PCR cycles to identify the
PCR products, which have specific melting temperatures. The method was used to simultaneously
detect Salmonella and L. monocytogenes employing specific genes for each group. After an overnight
LAMP—loop-mediated isothermal amplification
Rep-PCR—surveys repetitive sequences only
ERIC—enterobacterial repetitive intergenic concensus
Molecular Beacon—uses fluorescently labeled oligonucleotides in a qPCR format
QC-PCR—quantitative competitive
VNTR—variable number of tandem repeats (minisatellites)
Real-time PCR (qPCR, RTi-PCR)—PCR results in real time. TaqMan is a fluorogenic 5
nuclease
assay that produces results in real time.
A few applications of some of the above are summarized below.
FISH (fluorescent in situ hybridization) is an RNA probe method that targets 23S rRNA. To detect
Salmonella spp. in foods, two established oligonucleotide probes (Sal-1 and Sal-3) and a newly
constructed one were used. The probes were labeled with Cy3 (indocarboxyanine) and applied to
cultures of whole cells on slides.
55 Fluorescence occurred when the probes hybridized with the 23S
rRNA of target organisms (16S RNA sequences are more often used for bacteria). After slides were
prepared, they were counter-stained with DAPI ([4
, 6
-diamidino-2-phenylindole; a non-intercalating,
DNA-specific stain that fluoresces blue or bluish-white when bound to DNA and excited with light of
wavelength 365 nm),
109 and fluorescence was detected by epifluorescence microscopy. When FISH
was compared to a culture method for detecting Salmonella spp. in 18 different foods, up to 56
were found positive by at least one of the three FISH probes but only 30 of the 225 samples were
found positive by the culture method.
55 Overall, FISH detected salmonellae in 64% of the naturally
contaminated samples while culture methods detected in 13%. Of the 52 tested Salmonella serovars,
FISH hybridized with all 52. FISH was negative when tested on 46 non-salmonellae strains of 22
Enterobacteriaceae species, and on 14 species of 12 non-Enterobacteriaceae. Living, dead, and VBNC
(viable but nonculturable cells) could be detected. Among viable cells, those exposed to unfavorable
environmental conditions were detectable even though they may have been nonculturable. In this
study, investigators could detect salmonellae in food specimens 2–3 days sooner than by culturing.
Although it is unclear as to the minimum number of cells needed for a positive FISH response, this
number is probably >10
4 cfu/ml since it is a microscopic method.
LAMP is an in situ DNA amplification technique for the microscopic detection of bacteria. It is
similar to FISH in that they are microscope slide methods that emit fluorescence from Cy3- or DAPIstained cells. LAMP employs a low molecular weight DNA polymerase that enters whole cells; a
reaction temperature of 63
◦ C that makes it possible to use fluorescent antibody (FA) for simultaneous
cell detection; and generates large tandem repeats that prevent amplicons from leaking out of cells.
134
After DNA amplification using primers for the E. coli 0157:H7 stx 2 gene, the identification of these
cells was possible with FA labeling.
qPCR is a method whereby amplified gene products are detected by using fluorescence probes
during PCR cycling, and results can be obtained in 30–90 minutes. A number of fluorescent probes are
used such as SYBR Green I and fluorescence resonance energy transfer (FRET). These probes allow
the monitoring of the complete process, and they obviate the need for electrophoresis of end products.
FRET is a short oligonucleotide that is complementary to one DNA strand.
215 When this method was
employed to detect Giardia lamblia (the β-giardin gene was targeted) and Cryptosporidium parvum
(COWP gene was targeted), one cyst of the former and 100 cysts of the latter could be detected in
water and sewage samples.
74
Using SYBR Green I, melting curve analyses were made at the end of PCR cycles to identify the
PCR products, which have specific melting temperatures. The method was used to simultaneously
detect Salmonella and L. monocytogenes employing specific genes for each group. After an overnight