17.9.
34
AOAC Official Method 2007.02
Salmonella spp. in Select Foods
Ò
GeneQuence Salmonella
DNA Hybridization with 24 H Enrichment
First Action 2007
Final Action 2010
(Applicable to the detection of Salmonella spp. in raw turkey,
dried, liquid, and liquid frozen pasteurized eggs, milk chocolate, and
dry pet food.)
See Table 2007.02 for the results of the interlaboratory study
supporting acceptance of the method.
Caution: Reagents are for laboratory use only. Use of this test
should be restricted to personnel with appropriate
laboratory training in microbiology. Handle and
dispose of enrichment cultures as potentially infectious
material. Use either a disposable pipet and pipeting
bulb or micropipet with disposable tips to make all
pipeting transfers. Do not mouth pipet. The preferred
method for disposal of contaminated materials,
including cultures, pipets, etc., is autoclaving. Stop
solution contains 4 N sulfuric acid. Hybridization
solution contains formamide. Avoid contact with skin
and mucous membranes.
A. Principle
The GeneQuence Salmonella assay is a sandwich DNA
hybridization (DNAH) assay for detection of Salmonella spp. in
select foods. Following culture enrichment of test samples, bacteria
present in the culture are lysed to release ribosomal RNA (rRNA).
Horseradish peroxidase (HRP)-labeled detector probes and
polydeoxyadenylic acid (dA)-labeled capture probes hybridize to
Salmonella-specific sequences of the rRNA. The dA tail on the
capture probe facilitates binding of the hybridized complex to a
polydeoxythymidylic acid (dT)-coated microwell. Unbound probe
is removed by washing, and a chromogenic substrate of HRP is
added. After color development, the reaction is stopped by the
addition of acid, and absorbance at 450 nm is determined using a
photometer. Absorbance in excess of a threshold value indicates the
presumptive presence of Salmonella spp. in the sample. A positive
result must be confirmed by standard culture procedures such as
those described in the U.S. Department of Agriculture's Food Safety
and Inspection Service [USDA-FSIS; U.S. Department of
Agriculture-Food Safety Inspection Service (2004) Microbiology
Laboratory Guidebook, Ch. 4, http://www.fsis.usda.gov/PDF/MLG_
4_03.pdf] and the U.S. Food and Drug Administration's
Bacteriological Analytical Manual [FDA/BAM; U.S. Food and
Drug Administration (2003) Bacteriological Analytical Manual
Online, Ch. 5, http://www.cfsan.fda.gov/~ebam/bam-5.html]
reference methods.
Probes used in this test are not reactive with serovars of
Salmonella bongori.
B. Apparatus
Items (a)–(k) are available as GeneQuence Salmonella from
Neogen Corp. (Lansing, MI, USA; www.neogen.com). Each kit
contains sufficient materials and reagents for 100 tests. Store
reagents 1a, 2, 3, and 5 at 2–8°C. Other reagents may be stored at
2–25°C. Alternatively, the entire kit may be stored at 2–8°C.
Reagents from different kit lots should not be interchanged.
GeneQuence Salmonella reagents are not interchangeable with
other GeneQuence assay reagents. Reagents should not be used
beyond their expiration dates.
(a) Lysis reagent concentrate.—Reagent 1a, contains
proteinase K.
(b) Lysis reagent buffer.—Reagent 1b, contains Tris, pH 7.4,
disodium EDTA, n-lauryl sarcosine, and bromphenol blue.
(c) Hybridization solution.—Reagent 2, contains formamide.
(d) Salmonella probe solution.—Reagent 3, contains
HRP-labeled Salmonella-specific oligonucleotide probe and
p o l y d e o x y a d e n y l i c a c i d - l a b e l e d S a l m o n e l l a s p e c i f ic
oligonucleotide probe in Tris, pH 7.0, and cresol red.
(e) Wash solution, 20X concentrate.—Reagent 4, contains Tris,
disodium EDTA, NaCl, and Tween-20.
(f) Substrate-chromogen solution.—Reagent 5, contains urea
peroxide and tetramethylbenzidine.
(g) Stop solution.—Reagent 6, contains 4 N sulfuric acid.
(h) Positive control.—Contains formaldehy de-killed
S. Typhimurium.
(i) Negative control.—Contains formaldehy de-killed
Citrobacter freundii.
(j) Microwell plate.—96 wells in divisible strips (coated with
polydeoxythymidylic acid).
(k) Hybridization/probe mixture chart.
(l) High-speed blender.
(m) StomacherTM.
(n) Test tubes.—Glass, 12 ´ 75 mm.
(o) Test tubes or culture tubes.—50 mL, sterile.
(p) Micropipet and tips.—To dispense 100, 150, and 400 mL
volumes.
(q) 8-Channel pipettor and tips.—To dispense 50, 125, and
150 mL volumes.
(r) Repeater pipettor and syringe tips.—To dispense 100 mL
volume, optional.
(s) Incubator.—35 ± 1°C.
(t) Water bath or incubator.—42 ± 0.2°C.
(u) Water bath or heater block.—65 ± 1°C.
(v) Heater block.—45 ± 1°C, with insert for microtiter plate and
cover.
(w) Microwell plate or strip washing device.—8-Well
orientation, with vacuum source.
(x) Microwell plate or strip reader.—450 nm with discrimination
of 0.01 absorbance unit.
C. Media and Reagents
(a) Pre-enrichment media.—As appropriate for food type, per
FDA/BAM [U.S. Food and Drug Administration (2003)
Bacteriological Analytical Manual Online, Ch. 5,
http://www.cfsan.fda.gov/~ebam/bam-5.html; available from
Acumedia, Lansing, MI, USA, and other suppliers].
(b) Buffered peptone water (BPW).—Available from Acumedia
and other suppliers.
(c) Rappaport-Vassiliadis broth (RV).—Available from
Acumedia and other suppliers.
(d) Gram-negative broth (GN).—Available from Acumedia and
other suppliers.
(e) Brain heart infusion broth (BHI).—Available from
Acumedia and other suppliers.
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 Table 2007.02. Interlaboratory study results for the detection of Salmonella spp. in foods by the GeneQuence Salmonella assay
DNAH DNAH
Total Ref. False False
a b c d 2e f g h i
Food type Serovar Lev el MPN/g s am pl es Presumptive Confirmed m e th o d c S e n s i ti v i ty , % neg. , % S p e c i fi c i ty , % pos . , %
Raw ground turkey Group E Lot 1 11 78 73 73 76 0 .6 0 9 6 .1 3 .9 — —
Group E Lot 2 1 .4 9 78 75 74 78 2 .3 1 9 4 .9 5 .1 — —
Raw ground beef 1 S. Newport High 0 .4 3 71 55 54 63 3 .1 1 8 5 .7 1 4 .3 — —
Low 0 .0 7 4 72 59 58 65 2 .0 1 8 9 .2 1 0 .8 — —
Control < 0 .0 3 71 4 2 4 0 .1 7 5 0 .0 5 0 .0 — —

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Raw ground beef 2 S. Newport High 0 .4 3 66 53 51 58 1 .9 0 8 7 .9 1 2 .1 — —
Low < 0 .0 3 66 15 14 29 6 .7 6 4 8 .3 5 1 .7 — —
Control < 0 .0 3 66 0 0 0 — — — 1 0 0 .0 0 .0
Dried whole egg S. Enteritidis High 0 .2 1 66 58 58 61 0 .3 4 9 5 .1 4 .9 — —
Low 0 .1 4 66 41 41 43 0 .0 3 9 5 .3 4 .7 — —
Control < 0 .0 3 66 8 6 6 0 .0 9 1 0 0 .0 0 .0 — —
M i l k c h o c o l a te 1 S. Senftenberg High > 11 72 72 72 72 — 1 0 0 .0 0 .0 — —
Low > 11 72 72 72 72 — 1 0 0 .0 0 .0 — —
Control < 0 .0 3 72 8 0 0 — — — 8 8 .9 1 1 .1
M i l k c h o c o l a te 2 S. Senftenberg High 0 .1 5 66 51 50 52 0 .0 4 9 6 .2 3 .8 — —
Low 0 .0 9 2 66 23 23 25 0 .0 3 9 2 .0 8 .0 — —
Control < 0 .0 3 66 3 0 0 — — — 9 5 .5 4 .5
W a l n u ts S. Typhimurium High > 11 72 0 0 64 1 1 1 .6 0 .0 1 0 0 .0 — —
Low 0 .0 7 4 72 1 0 32 3 8 .6 0 .0 1 0 0 .0 — —
Control < 0 .0 3 72 0 0 0 — — — 1 0 0 .0 0 .0
Dry pet food S. Thompson High 0 .0 3 6 60 39 38 40 0 .0 4 9 5 .0 5 .0 — —
Low 0 .0 3 60 13 13 14 0 .0 0 9 2 .9 7 .1 — —
Control < 0 .0 3 60 0 0 0 — — — 1 0 0 .0 0 .0
a
MPN = Most probable number of colony-forming units (CFU)/g food.
b
Number of DNAH assay positive samples not considering subsequent confirmation.
c
Number of DNAH assay positive samples subsequently confirmed.
d
Number of samples positive by reference method (USDA-FSIS method for raw ground turkey, raw ground beef, and dried whole egg; FDA/BAM method for milk chocolate, walnuts, and dry pet food).
e 2 2
c is defined as N*{[a*d – b*c] – N/2} /[(a + b)(a + c)(c + d)(b + d)], where a = samples positive by test method, b = samples positive by reference method, c = samples negative by test method, d = samples
negative by reference method, N = total samples tested by both methods.
f
Sensitivity rate is defined as the number of DNAH confirmed positive test results divided by the number of reference method positive test results, expressed as a percentage.
g
False-negative rate = 100 – sensitivity rate.
h
Specificity rate is defined as the number of DNAH assay negative samples divided by the number of negative samples, expressed as a percentage. Calculated only for control levels with no confirmed positive
results.
i
False-positive rate = 100 – specificity rate.
D. Enrichment
Note: All enrichment media should be prewarmed to room
temperature before use.
(a) For dried, liquid, and liquid frozen pasteurized eggs.—(1)
Homogenize (Stomacher) sample in 9 volumes BPW. [Note: The
standard sample size as specified by USDA-FSIS is 100 g; U.S.
Department of Agriculture-Food Safety Inspection Service (2004)
Microbiology Laboratory Guidebook, Ch. 4,
http://www.fsis.usda.gov/PDF/MLG_4_03.pdf.] For dried egg
samples, add BPW to the sample gradually, and mix to produce a
homogeneous suspension free of lumps. Incubate 18–20 h at 35 ±
1°C.
(2) Remove BPW culture from incubation, and mix well.
Transfer 10 mL BPW culture to 10 mL double-strength GN broth in
50 mL tube. Incubate 6–7 h at 35 ± 1°C.
(3) Remove GN culture from incubation and mix. Perform
GeneQuence Salmonella assay using 0.4 mL GN culture. Save GN
culture at 2–8°C for possible confirmation.
(b) For dry pet food.—(1) Blend sample in 9 volumes lactose
broth in accordance with FDA/BAM guidelines [U.S. Food and
Drug Administration (2003) Bacteriological Analytical Manual
Online, Ch. 5, http://www.cfsan.fda.gov/~ebam/bam-5.html].
Incubate 18–20 h at 35 ± 1°C.
(2) Remove pre-enrichment culture from incubation, and mix
well. Transfer 10 mL pre-enrichment culture to 10 mL
double-strength GN broth in 50 mL tube. Incubate 6–7 h at
35 ± 1°C.
(3) Remove GN culture from incubation, and mix. Perform
GeneQuence Salmonella assay using 0.4 mL GN culture. Save GN
culture at 2–8°C for possible confirmation.
(c) For milk chocolate.—(1) Blend sample in 9 volumes skim
milk medium with brilliant green in accordance with FDA/BAM
guidelines [U.S. Food and Drug Administration (2003)
Bacteriological Analytical Manual Online, Ch. 5,
http://www.cfsan.fda.gov/~ebam/bam-5.html]. Incubate 22–24 h at
35 ± 1°C.
(2) Remove pre-enrichment culture from incubation, and mix
well. Transfer 10 mL pre-enrichment culture to 10 mL
double-strength BHI broth in 50 mL tube. Incubate 6–7 h at
35 ± 1°C.
(3) Remove BHI culture from incubation, and mix. Perform
GeneQuence Salmonella assay using 0.4 mL BHI culture. Save BHI
culture at 2–8°C for possible confirmation.
(d) For raw poultry.—(1) Homogenize (stomacher) sample in 9
volumes RV broth. Incubate 18–20 h at 42 ± 0.2°C.
(2) Remove RV culture from incubation and mix well. Transfer
1 mL RV culture to 10 mL GN broth. Incubate 6–7 h at 35 ± 1°C.
(3) Remove GN culture from incubation and mix. Perform
GeneQuence Salmonella assay using 0.4 mL GN culture. Save GN
culture at 2–8°C for possible confirmation.
Note: Samples must be tested when the final GN or BHI
incubation period is complete. Do not hold cultures for testing at a
later time.
E. General Preparation
General notes.—This assay should be performed in a normal
laboratory environment with respect to humidity, lighting, etc. Steps
requiring room temperature incubation should be performed at
18–30°C. Kit reagents that have been refrigerated should be
equilibrated to room temperature before use but should not be left
out unrefrigerated for long periods of time (more than 2 h).
Prior to starting the assay.—(1) Turn on the water bath or heater
block and adjust to 65 ± 1°C. Water bath should be filled to a level of
ca 1.5 in. Heater block wells should be filled about 1/3 with
deionized water.
(2) To reagent bottle 1a (lysis reagent concentrate), add 6 mL
solution 1b (lysis reagent buffer). Dissolve contents by gentle
swirling and place the bottle on ice.
Note: Lysis reagent is stable in the reconstituted form for 60 days
when stored at –20°C. To thaw, place bottle at room temperature.
When thawed, place on ice (freeze-thawing does not adversely
affect the reconstituted reagent).
(3) For each sample to be tested, label a 12 ´ 75 mm glass test
tube with the appropriate sample designation and place in a rack.
Include tubes for one positive control and one negative control per
experimental run.
(4) Prepare 1X wash solution. Dilute solution 4 (20X wash
solution concentrate) by mixing contents of bottle (25 mL) with
475 mL distilled or deionized water. Fill buffer reservoir of plate
washing device.
Note: 1X wash solution can be stored in a closed bottle at room
temperature for up to 60 days.
(5) Prepare a mixture of solution 2 (hybridization solution) and
solution 3 (Salmonella probe solution) in an appropriate size plastic
or glass container in a 4:1 proportion to achieve a sufficient amount
for the number of samples to be tested. Use the formula below or
refer to the mixing chart available from Neogen Corp.
Volume solution 2 = [(N ´ 0.1) + 1.6] mL
Volume solution 3 = [(N ´ 0.025) + 0.4] mL
where N is the number of samples to be tested including controls.
Note: The above formula provides for an excess volume of
2.0 mL; a different excess volume may be appropriate depending on
the type of container used to prepare the mixture and the type of
pipetting device used.
(6) Place the appropriate number of coated microwells in the
plate frame, filling the frame left to right and front to back in rows of
8. Include wells for the reagent blank, negative control, and positive
control. Avoid touching the bottoms of the wells. If the last row has
fewer than 8 wells, fill in the row as necessary with the special
uncoated wells (colored bottoms) provided.
F. Analysis
(1) Mix the sample cultures (GN or BHI cultures). Add 0.4 mL of
each GN or BHI culture to the appropriate tubes. Shake the positive
control and negative control solutions by inverting the bottles
several times, and add 0.4 mL of each control to the appropriate
tubes.
(2) Add 0.1 mL solution 1 (reconstituted lysis reagent) to each
tube. Shake the rack of tubes by hand for 5 s. The resulting solution
should be blue. If any tubes are not blue, check for proper reagent
addition. Incubate the rack of tubes in the 65°C water bath or heater
block for 5 min.
(3) Remove the rack of tubes from the 65°C water bath or heater
block. Transfer 0.15 mL of each lysed sample, including the
controls, to the designated microwell. The first well should be
reserved for the reagent blank and receives no sample. The second
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well should be used for the negative control, and the third well for
the positive control.
(4) Using an 8-channel pipettor, add 0.125 mL premixed solution
2/3 (hybridization/probe solution) prepared in E(5) to each well
except the reagent blank well. Mix the contents of the wells 5–10
times using the pipettor.
(5) Incubate the plate on the covered heater block at 45°C for 1 h.
(6) Wash the wells 5 times at room temperature using a plate or
strip washing device. After the last wash, remove residual liquid by
inverting the plate and tapping it onto absorbent paper. Hold the
plate by gently squeezing on the sides of the frame to keep the strips
in place.
(7) Add 0.15 mL solution 5 (substrate-chromogen solution) to
each well, including the reagent blank well. Incubate the plate at
room temperature for 20 min.
(8) Add 0.05 mL solution 6 (stop solution) to each well, including
the blank well.
(9) Read absorbance at 450 nm using a plate or strip reader
according to the manufacturer’s instructions. Blank on the reagent
blank (do not blank on air).
G. Results
(a) Control values.—The absorbance value for the negative
control must be £0.15. Otherwise, the assay is invalid and should be
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repeated. The absorbance value for the positive control must be
³1.00. Otherwise, the assay is invalid and should be repeated.
(b) Negative criterion.—Assays producing an absorbance value
<0.10 indicate the absence of Salmonella spp. in the test sample.
(c) Positive criterion.—Assays producing an absorbance value
³0.10 indicate the presence of Salmonella spp. in the test sample.
These samples should be confirmed by standard culture procedures.
H. Confirmation of Positive Results
For samples which produce positive hybridization assays, the
result should be confirmed by streaking from the GN or BHI cultures
and/or the selective enrichment cultures to selective/differential
plating media (Hektoen Enteric Agar, Xylose Lysine Deoxycholate
Agar, and Bismuth Sulfite Agar are recommended) and continuing
with biochemical and serological identification of presumptive
Salmonella spp. isolates using standard procedures [U.S.
Department of Agriculture-Food Safety Inspection Service (2004)
Microbiology Laboratory Guidebook, Ch. 4,
http://www.fsis.usda.gov/PDF/MLG_4_03.pdf; U.S. Food and
Drug Administration (2003) Bacteriological Analytical Manual
Online, Ch. 5, http://www.cfsan.fda.gov/~ebam/bam-5.html].
Reference: J. AOAC Int. 90, 738(2007).