THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO.
2, 2006 417
FOOD BIOLOGICAL CONTAMINANTS
Singlepath Salmonella ®
Performance-Tested MethodSM 060401
Abstract
Singlepath® Salmonella is an immunochromatographic
(lateral flow) assay for the presumptive qualitative detection
of Salmonella spp. in food. The AOAC Performance-Tested
MethodSM study evaluated Singlepath Salmonella as an
effective method for the detection of Salmonella spp. in the
following selected foods: dried skimmed milk, black pepper,
dried pet food, desiccated coconut, cooked peeled frozen
prawns, raw ground beef, and raw ground turkey. When the
foods were inoculated with Salmonella spp. at levels ranging
from low [0.23–1.08 colony forming units (CFU)/25 g] to
high (2.3–6.0 CFU/25 g), a Chi-square value of 0.9 indicated
that there was no significant difference between Singlepath
Salmonella and the ISO 6579:2002 reference method.
Singlepath Salmonella gave a false-positive rate of 7.3% and a
false-negative rate of 2.5%. For the inclusivity study, all
105 Salmonella serovars reacted with Singlepath Salmonella.
For the exclusivity study, 58 non-Salmonella spp. were tested.
There were no cross-reactions with Singlepath Salmonella
from these strains.
1 Scope of Method
(1.1) Target organisms.—Salmonella spp.
(1.2) Matrixes.—Dried skimmed milk, black pepper,
dried pet food, desiccated coconut, cooked peeled frozen
prawns, raw ground beef, and raw ground turkey.
(1.3) Summary of validated performance
claims.—SinglepathÒ Salmonella enables the detection of
Salmonella spp. at low contamination levels (1–10 CFU/25 g)
from the following selected foods: dried skimmed milk, black
pepper, dried pet food, desiccated coconut, cooked peeled frozen
prawns, raw ground beef, and raw ground turkey. Singlepath
Salmonella shows equal sensitivity (occurrence of false negatives)
and specificity (occurrence of false positives) with ISO
6579:2002 (1) for detection of Salmonella spp.
2 Participants
METHOD AUTHORS
LISA THOMPSON and CHARLOTTE LINDHARDT
Merck Chemicals Ltd., Newport Technical Centre,
5 The Courtyard, Imperial Park, Newport NP10 8UL, Wales,
United Kingdom
Received March 31, 2005. Accepted by AH September 6, 2005.
Corresponding authors' e-mail: lisa.thompson@merckchem.co.uk;
charlotte.lindhardt@merckchem.co.uk
SUBMITTING COMPANY
Merck KGaA, Life Science and Analytics/Research and
Development/Assay Development and Microbiology
(LSA/R&D/AD&M) Frankfurter Strasse 250, 64271
Darmstadt, Germany
INDEPENDENT LABORATORY
r-tech laboratories, c/o Land O’Lakes, 1150 W. County Rd F,
Dock #2, Arden Hills, MN 55112; Analytical Laboratory,
PO Box 64101, St. Paul, MN 55164-0101
REVIEWERS
JOSEPH ODUMERU
University of Guelph, Laboratory Services Division,
Department of Sciences, 95, Stone Rd West (Laboratory),
Guelph, Ontario, N1H 8J7 Canada
JOSEPH D. EIFERT
Virginia Polytechnic Institute and State University,
Department of Food Science and Technology (0418),
Blacksburg, VA 24061
3 Introduction
3.1 Principle
Singlepath Salmonella (Cat. No. 1.04140.0001) is an
immunochromatographic rapid test based on gold-labeled
antibodies. The test device has a circular sample port and an
oval shaped test (T) and control (C) window.
After enrichment in buffered peptone water (BPW) and
Rappaport-Vassiliadis soy (RVS) broth according to
ISO 6579:2002 (1) or equivalent enrichment methods such as
USDA-FSIS Microbiology Laboratory Guidebook (MLG)
4.02 (2), the selective enrichment sample can be tested, prior
to plating, for isolated colonies. The selective enrichment
sample is boiled, cooled to room temperature, and applied to
the nitrocellulose membrane via the circular sample port. The
sample is absorbed through the pad to the reaction zone
containing colloidal, gold-labeled antibodies specific to
Salmonella spp. Any Salmonella antigen present, complexes
with the gold-labeled antibody and migrates along the
membrane until it encounters the binding zone in the test (T)
area. The binding zone (T) contains another anti-Salmonella
antibody, which immobilizes any Salmonella antigen-antibody
complex present. Due to the gold-labeling, a distinct red line is
then formed. The rest of the sample continues to migrate to a
second binding reagent zone within the control (C) zone, and
also forms a second distinct red line (positive control). At the
control zone, an antibody directed against the gold-labeled
418 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006
antibody is immobilized onto the membrane. Regardless of
whether any Salmonella antigen is present or not, this distinct
red line is always formed in the control (C) zone within
20 min, thus ensuring the test is working correctly.
When Salmonella spp. are present in the sample, the
formation of 2 distinct red lines is observed. Only 1 red line
(control) is observed when no Salmonella spp. are present (see
Figure 1).
3.2 Summary of Results
The following foods were inoculated with Salmonella
spp.: dried skimmed milk, black pepper, dried pet food,
desiccated coconut, cooked peeled frozen prawns, raw ground
beef, and raw ground turkey. When these foods were
inoculated with Salmonella spp. at levels ranging from low
(0.23–1.08 CFU/25 g) to high (2.3–6.0 CFU/25 g), a
Chi-square value of 0.9 indicated that there was no significant
difference between Singlepath Salmonella and the ISO
6579:2002 reference method (1). Singlepath Salmonella gave
a false-positive rate of 7.3% and a false-negative rate of 2.5%.
For the inclusivity study, all 105 Salmonella serovars reacted
with Singlepath Salmonella. For the exclusivity study,
58 non-Salmonella spp. were tested. There were no
cross-reactions with Singlepath Salmonella from these strains.
4 Materials and Method
4.1 Test Kit Information
(4.1.1) Kit name.—Singlepath Salmonella Lateral Flow
assay.
(4.1.2) Cat. No.—1.04140.0001.
(4.1.3) Ordering information.
(4.1.3.1) Inside the United States.—EMD Chemicals,
480 S. Democrat Rd, Gibbstown, NJ 08027-1297, Tel:
800-222-0342 or 856-423-6300, Fax: 856-423-4389,
e-mail: emdinfo@emdchemicals.com, Website:
www.emdchemicals.com.
Figure 1. Reaction patterns obtained with Singlepath
Salmonella.
(4.1.3.2) Europe/international.—Merck KGaA, 64271
Darmstadt, Germany, Tel: +49-6151-72-0, Fax:
+49-6151-72-2000, Website: www.merck.de.
(4.1.4) Media and reagents.
(4.1.4.1) BPW.—e.g., Merck KGaA, Cat. No.
1.07228.0500.
(4.1.4.2) RVS broth.—e.g., Merck KGaA, Cat. No.
1.07700.0500.
(4.1.4.3) Singlepath Salmonella.—Merck KGaA, Cat.
No. 1.04140.0001.
4.2 Additional Supplies and Reagents
(4.2.1) Lactose monohydrate.—e.g., Merck KGaA, Cat.
No. 101394S.
(4.2.2) Muller-Kauffmann tetrathionate novobiocin
(MKTTn) broth.—According to ISO 6579:2002 (1), e.g.,
Merck KGaA, Cat. No. 1.05878.0500.
(4.2.3) Potassium iodide.—e.g., Merck KGaA, Cat. No.
29631 2C.
(4.2.4) Iodine.—e.g., Merck KGaA, Cat. No. 10135 3J.
(4.2.5) Brilliant green.—e.g., Merck KGaA, Cat. No.
34015 4J.
(4.2.6) Xylose lysine desoxycholate agar
(XLD).—According to ISO 6579:2002, e.g., Merck KGaA,
Cat. No. 1.05287.0500.
(4.2.7) Hektoen enteric agar (HE).—e.g., Merck KGaA,
Cat. No. 1.11681.0500.
(4.2.8) Bismuth sulfite agar.—e.g., Merck KGaA, Cat.
No. 1.05418.0500.
(4.2.9) Triple sugar/iron agar.—Merck KGaA, Cat. No.
1.03915.0500.
(4.2.10) API 20E Enterobacteriaceae Identification
Kit.—bioMJrieux, Marcy-l’Etoile, France, Cat. Nos. 20100
(strips) and 20120 (reagents).
(4.2.11) Gram-negative identification (GNI; Vitek)
biochemical identification system (external validation
study).—bioMJrieux.
(4.2.12) Maximum recovery diluent.—e.g., Merck KGaA,
Cat. No. 1.12535.0500.
(4.2.13) Nutrient agar.—e.g., Merck KGaA, Cat. No.
1.05450.0500.
(4.2.14) Salmonella O and H antisera for serotype
confirmation.—Prolab Diagnostics, Cheshire, UK; Sifin,
Berlin, Germany.
(4.2.15) Sterile physiological saline solution.—For
serotyping and preparation of API 20E inocula.
(4.2.16) Distilled/deionized water.—For media
preparation.
(4.2.17) Sterile disposable containers.—For enrichments.
(4.2.18) Disposable plastic transfer pipets.
(4.2.19) Disposable heat-stable polypropylene tubes for
boiling samples.
(4.2.20) Sterile disposable tips for micropipets.
(4.2.21) Sterile Stomacher bags with filters.—For use
with Stomacher 400 Circulator (Seward 400 Circulator,
Seward, London, UK).
(4.2.22) Sterile, disposable inoculation needles.
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 419
(4.2.23) Sterile, disposable 10 mL inoculation loops.
4.3 Apparatus
(4.3.1) Stomacher blender.—Seward 400 Circulator.
(4.3.2) Static incubator(s).—Capable of maintaining
temperatures, according to ISO 6579:2002 reference
method (1), of 37 ± 1°C and 41.5 ± 1°C.
(4.3.3) Autoclave.—For sterilizing media at 121 and
115°C for 15 min and for sterilizing enrichments and test kits
after use.
(4.3.4) Conical glass flasks (2 L).—For pre-enrichment of
100 g samples (for MPN series).
(4.3.5) Waterbath/heating block.—For boiling samples.
(4.3.6) Open pan weighing balance.—Capable of
weighing 0.1–25 g of sample matrix.
(4.3.7) Micropipets.—Capable of accurately dispensing
100, 160, and 1000 mL sample.
(4.3.8) Plastic bottles and drums.—For mixing spiked
food samples.
4.4 Standard Reference Materials
(4.4.1) Bacterial strains for inclusivity and exclusivity
testing were obtained from the following sources: American
Type Culture Collection (ATCC), Manassas, VA; National
Collection of Type Cultures (NCTC), Health Protection
Agency (HPA), Central Public Health Laboratory, London,
UK; Robert-Koch Institute, Berlin-Wedding, Germany;
National Reference Center for Salmonellae, Hamburg,
Germany; Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH (DSMZ), German Collection of
Microorganisms and Cell Cultures, Braunschweig, Germany;
Merck Chemicals, In-House Culture Collection, Newport,
Wales, UK.
(4.4.2) The identity of all strains from Merck Chemicals
In-House Culture Collection were confirmed by biochemical
testing using bioMJrieux API 20E identification system and
by full serotyping of somatic (O) and flagellar antigens
(H1 and H2).
4.5 Standard Solutions
Not applicable.
4.6 General Preparation
(4.6.1) Prepare all media and reagents according to
manufacturer’s instructions prior to analysis.
(4.6.2) Sterilize 2 L flasks for most probable number
(MPN) series prior to use.
(4.6.3) Turn on incubators and set to 37 ± 1°C and
41.5 ± 1°C.
(4.6.4) Turn on water bath (to boiling) or heating block
(set to 95°C) for boiling samples.
(4.6.5) Remove the required number of Singlepath
Salmonella devices from 4°C storage and allow to equilibriate
to room temperature (18–26°C) for at least 30 min in foil
pouches before use.
4.7 Sample Preparation
(4.7.1) Samples should be collected according to standard
laboratory techniques, such as those described in the
Bacteriological Analytical Manual Online (BAM; 3),
MLG (2), or the ISO standard specific for the product
concerned.
(4.7.2) Samples should be enriched according to ISO
6579:2002 (1).
(4.7.3) Transfer 1–2 mL RVS culture to an appropriate
(polypropylene) tube. Cover with a loose-fitting cap.
(4.7.4) Place tubes in a boiling water bath or heating block
set at 95°C for 15 min.
(4.7.5) Remove and allow cooling to room temperature
(18–26°C), prior to use.
4.8 Analysis
(4.8.1) Remove foil pouches from the required number
of Singlepath Salmonella devices. Place test device(s) on a
flat surface and label with appropriate sample
identification. (Note: Perform tests within a period of 2 h
after opening.)
(4.8.2) Using a micropipet and disposable pipet tip,
dispense 160 mL of the sample into the circular sample port on
the test device.
(4.8.3) Observe the test result 20 min after applying the
sample to the device.
4.9 Interpretation and Test Result Report
(4.9.1) The test can be regarded as working correctly if a
distinct red line appears in the control zone (C) within 20 min.
(4.9.2) A sample can be considered positive if at or before
20 min, red lines appear on both test (T) and control (C) zones.
(4.9.3) A sample can be considered negative if no red line
appears in the test (T) zone but does appear distinctly in the
control (C) zone 20 min after application of sample to the
device.
5 Safety Precautions
(5.1) Cultures and samples are (potentially) contaminated
with pathogens (Biosafety Level 2/ACDP class 2).
(5.2) All work with samples, live sample enrichments, and
pure cultures must be carried out aseptically to avoid
cross-contamination among samples or contamination of
laboratory personnel.
(5.3) All cultures and samples must be sterilized by
autoclaving or chemical treatment prior to disposal. Test
devices run with boiled samples need not be sterilized prior to
disposal.
6 Summary of Results
Internal validation studies were conducted in the
laboratories of Merck Chemicals Ltd.
420 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

6.1 Ruggedness Testing
The effects of perturbations in 3 method parameters were
investigated on 2 different days: (1) Sample time in boiling
water bath (0, 15, and 25 min); (2) time for sample result
observation (20 and ± –5 min); and (3) sample volume for
assay (160 ± –10 mL).
(6.1.1) Methodology.
(6.1.1.1) Sample preparation for all 3 method parameters.
(6.1.1.1.1) For Day 1, 2 positive strains, S. Enteritidis
(NCTC 5188) and S. Typhimurium (ATCC 14028) were
cultured from cryobeads to BPW, enriched at 37°C for 20 h,
inoculated to RVS broth at 1%, and enriched 41.5°C for 24 h.
(6.1.1.1.2) Plate counts were performed on Nutrient Agar
to determine CFU/mL. The positive strains were diluted to
105 CFU/mL in RVS, for analysis to be within 1 log of the
limit of detection of the kit for these strains. This dilution was
used to analyze all 3 method parameters.
(6.1.1.1.3) For Day 1, one negative strain, E. coli (ATCC
25922) was cultured from cryobeads to BPW and enriched at
37°C for 20 h. It was agreed with AOAC (AOAC Validation
Outline v4), not to culture E. coli into selective RVS, as it
would not be culturable to a cell density that could have an
effect on Singlepath Salmonella performance. Plate counts
were performed on Nutrient Agar to determine CFU/mL. The
negative strain was used for analysis undiluted in BPW.
(6.1.1.1.4) For Day 2, the 2 positive strains were prepared
by inoculating fresh RVS broth with 1% of the primary BPW
culture used to prepare Day 1 cultures, and incubating
overnight at 41.5°C. The negative strain, E. coli
(ATCC 25922), was prepared by inoculating fresh BPW with
the Day 1 E. coli culture, and incubating overnight at 37°C.
Fresh plate counts were performed and strains analyzed as for
Day 1.
(6.1.1.2) Sample time in boiling water bath (0, 15, and
25 min).
(6.1.1.2.1) Singlepath Salmonella kit insert recommends
preparing the sample for analysis by boiling in a water bath for
15 min.
(6.1.1.2.2) In this study, the 2 positive and 1 negative
strains were prepared for analysis by not boiling (0 min), and
boiling for 15 and 25 min.
(6.1.1.2.3) Boiled samples were allowed to cool to room
temperature (18–26°C) for at least 30 min.
(6.1.1.2.4) Samples containing 160 mL were applied to
tests and read at 20 min. Five replicates were tested per strain.
(6.1.1.2.5) This study was the first performed on each day,
to enable samples boiled for 15 min (standard Singlepath
protocol) to be used in the remaining 2 investigations.
(6.1.1.3) Time for sample result observation: 20 ± 5 min.
(6.1.1.3.1) Singlepath Salmonella kit insert recommends
reading the tests at or within 20 min of sample application.

Table 1. Singlepath® Salmonella ruggedness testing results: water bath boil; positive signal at control zone for all
tests
Water bath boil, min

Day 1 Day 2

0 15 25 0 15 25
a
Sample Replicate T T T Sample T T T

S. Enteritidis NCTC 5188 a + + + S. Enteritidis NCTC 5188 + + +

b + + + + + +
c + + + + + +
5 5
7.49 ´ 10 CFU/mL d + + + 8.40 ´ 10 CFU/mL + + +
e + + + + + +
S. Typhimurium ATCC
S. Typhimurium ATCC 14028 a + + + 14028 – + +
b + + + – + +
c + + + – + +
5 5
6.58 ´ 10 CFU/mL d + + + 9.85 ´ 10 CFU/mL – + +
e + + + – + +
E. coli ATCC 25922 a – – – E. coli ATCC 25922 – – –
b – – – – – –
c – – – – – –
8 8
3.13 ´ 10 CFU/mL d – – – 7.83 ´ 10 CFU/mL – – –
e – – – – – –

a
T = Test.
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 421

Table 2. Singlepath® Salmonella ruggedness testing results: sample result observation; positive signal at control
zone for all tests
Day 1 Day 2

Sample result observation, min Sample result observation, min

15 20 25 15 20 25
a
Sample Replicate T T T Sample T T T

S. Enteritidis NCTC 5188 a + + + S. Enteritidis NCTC 5188 + + +

b + + + + + +
5 5
7.49 ´ 10 CFU/mL (Day 1) c + + + 8.40 ´ 10 CFU/mL (Day 2) + + +
d + + + + + +
e + + + + + +
S. Typhimurium ATCC 14028 a + + + S. Typhimurium ATCC 14028 + + +
b + + + + + +
5 5
6.58 ´ 10 CFU/mL (Day 1) c + + + 9.85 ´ 10 CFU/mL (Day 2) + + +
d + + + + + +
e + + + + + +
E. coli ATCC 25922 a – – – E. coli ATCC 25922 – – –
b – – – – – –
8 8
3.13 ´ 10 CFU/mL (Day 1) c – – – 7.83 ´ 10 CFU/mL (Day 2) – – –
d – – – – – –
e – – – – – –

a
T = Test.

(6.1.1.3.2) In this study, 160 mL of 15 min boiled samples
of the 2 positive and 1 negative strains were applied to the
tests. Five replicates were tested per strain.
(6.1.1.3.3) Tests were read at 15, 20, and 25 min.
(6.1.1.4) Sample volume for assay: 160 ± 10 mL.
(6.1.1.4.1) Singlepath Salmonella kit insert recommends
applying about 160 mL sample to each test.
(6.1.1.4.2) In this study, 150, 160, and 170 mL of the
15 min boiled and samples of the 2 positive and 1 negative
strains were applied to each test. Five replicates were tested
per strain.
(6.1.1.4.3) Tests were read at 20 min.
(6.1.2) Results.—All results of the ruggedness study are
summarized in Tables 1–3.
(6.1.2.1) Sample time in boiling water bath (0, 15, and
25 min).
(6.1.2.1.1) On Day 1, when tests were read at 20 min,
Singlepath Salmonella detected both positive strains at all
3 boiling times, and the negative strain was negative at all
3 boiling times.
(6.1.2.1.2) On Day 2, S. Typhimurium was not detectable
by Singlepath Salmonella when unboiled (0 min boiling time).
(6.1.2.1.3) On Day 2, the S. Enteritidis strain was
detectable at all 3 boiling times and the negative strain was
negative at all 3 boiling times. Time for sample result
observation: 20 ± 5 min.
(6.1.2.2) Time for sample result observation: 20 ± 5 min.
(6.1.2.2.1) On Day 1 and Day 2, Singlepath Salmonella
detected both positive strains, when read at all 3 timepoints.
The negative strain was negative at all 3 timepoints.
(6.1.2.3) Sample volume for assay: 160 ± 10 mL.
(6.1.2.3.1) On Day 1 and 2, Singlepath Salmonella
detected both positive strains, when tested with all 3 sample
volumes. The negative strain was negative with all 3 sample
volumes.
6.2 Lot-to-Lot Testing
According to real time data available at the time this report
was submitted, Singlepath Salmonella had a shelf life of
1 year (already extended to 15 months). Three different lots
tested at 6, 9, and 14 months after manufacturing were tested
at the same time as Day 2 of the ruggedness study on
February 5, 2004.
(6.2.1) Methodology.
(6.2.1.1) The 2 positive and 1 negative strains prepared for
the ruggedness study Day 2 were also used in this lot-to-lot
study. Refer to 6.1.1.1.4 for sample preparation.
(6.2.1.2) The positive strains that had been diluted to
105 CFU/mL in RVS and the negative strain undiluted in BPW
422 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

Table 3. Singlepath® Salmonella ruggedness testing results: sample volume for assay; positive signal at control
zone for all tests
Day 1 Day 2

Sample volume for assay, mL Sample volume for assay, mL

150 160 170 150 160 170

a
Sample Replicate T T T Sample T T T

S. Enteritidis NCTC 5188 a + + + S. Enteritidis NCTC 5188 + + +

b + + + + + +
5 5
7.49 ´ 10 CFU/mL (Day 1) c + + + 8.40 ´ 10 CFU/mL (Day 2) + + +
d + + + + + +
e + + + + + +
S. Typhimurium ATCC 14028 a + + + S. Typhimurium ATCC 14028 + + +
b + + + + + +
5 5
6.58 ´ 10 CFU/mL (Day 1) c + + + 9.85 ´ 10 CFU/mL (Day 2) + + +
d + + + + + +
e + + + + + +
E. coli ATCC 25922 a – – – E. coli ATCC 25922 – – –
b – – – – – –
8 8
3.13 ´ 10 CFU/mL (Day 1) c – – – 7.83 ´ 10 CFU/mL (Day 2) – – –
d – – – – – –
e – – – – – –

a
T = Test.

were tested as boiled (for 15 min) samples on 3 different lots
of test kits: (1) 03226-016, manufactured 08/2003;
(2) 03133-015, manufactured 05/2003; and (3) 02329-005,
manufactured 12/2002.
(6.2.1.3) A 160 mL volume of each sample was applied to
tests from the 3 different lots. Five replicates were tested per
strain.
(6.2.1.4) Tests were read at 20 min.
(6.2.2) Results.—All results are shown in Table 4.
6.3 Inclusivity Testing
A total of 105 Salmonella serovars/strains of
Salmonella spp. were obtained from a combination of the
sources specified in the Standard Reference Materials,
Section 4.4 of this report. Strains from Merck Chemicals
In-House Culture Collection were confirmed before use by
the biochemical BioMJrieux API 20E identification system
and by the serotyping of somatic (O) and flagellar antigens
(H1 and H2).
(6.3.1) Methodology.
(6.3.1.1) Nonselective BPW was inoculated from
cryobead/liquid stocks with all strains, and enriched for
18 ± 2 h at 37 ± 1°C.
(6.3.1.2) A 0.1 mL portion of all pre-enrichments was
inoculated into 10 mL of the selective enrichment broth, RVS,
and enriched for 24 ± 3 h at 41.5 ± 1°C.
(6.3.1.3) All RVS enrichments were serially diluted in
0.9% saline, and plate counts on Nutrient Agar were set up.
The strains reached various cell densities during enrichment,
ranging from 105 to 108 CFU/mL.
(6.3.1.4) All undiluted RVS enrichments were boiled for
15 min, and allowed to cool to room temperature (18–26°C)
for at least 30 min.
(6.3.1.5) A 160 mL volume of boiled undiluted sample was
applied into the sample port of each Singlepath Salmonella
device.
(6.3.1.6) Results were recorded as positive or negative
after 20 min.
(6.3.1.7) For strains that gave strong positive test signals
with the undiluted sample, 1:10 and 1:100 dilutions were
made in RVS, boiled for 15 min, cooled, and tested with
Singlepath. This gave information on detection limits.
(6.3.2) Results.—All results obtained with the 105 strains
of Salmonella spp. are summarized in Table 5. As shown in
Table 5, all strains provided a positive signal at the test and
control zones of Singlepath Salmonella. The strength of the
test signal varies with serogroup. The detection limit varies
with serogroup from 104 to 108 CFU/mL.
6.4 Exclusivity Testing
The 58 strains not belonging to the genus Salmonella were
obtained from a combination of the sources specified in the
Standard Reference Materials, Section 4.4 of this report.
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 423

Table 4. Singlepath® Salmonella lot-to-lot consistency
study results: positive signal at control zone for all tests
Singlepath Salmonella lot No.
and results
03133-015 02329-005 03226-016
into the selective broth, RVS, these strains were not culturable
and did not produce cross-reactions.
(6.4.2.2) Five strains were positive when initially tested in
the nonselective broth, BPW, and were culturable in RVS, but
the RVS sample retested negative.

Boiled 6.5 Food Testing

a
Sample Replicate T T T (6.5.1) Methodology.—The purpose of the study was to
compare the Singlepath Salmonella assay to the ISO reference
S. Enteritidis a + + +
ATCC 14028 method, 6579:2002 (1) for detection of Salmonella spp. in
5 dried skimmed milk powder, black pepper, dried pet food (dog
8.40 ´ 10 CFU/mL b + + +
food), desiccated coconut, and cooked peeled frozen prawns.
c + + + All matrixes, except coconut, were obtained commercially
d + + + from local supermarkets. Approximately 2.5 kg desiccated
e + + + coconut, naturally contaminated with S. Java was donated by
Janet Corry, Department of Clinical Veterinary Science,
S. Typhimurium a + + +
NCTC 5188
University of Bristol, Bristol, UK. An overview of the
5 methodology is shown in Figure 2.
9.85 ´ 10 CFU/mL b + + +
(6.5.1.1) Spiking of products.—Cultures for spiking were
c + + + grown overnight in Nutrient Broth at 37 ± 1°C. Cultures were
d + + + streaked on XLD, HE, and Nutrient Agar for purity check and
e + + + serotype confirmation.
E. coli ATCC 25922 a – – –
(6.5.1.1.1) Spiking of dried products.—Dried inocula
8 were prepared by air-drying a small volume of fresh liquid
7.83 ´ 10 CFU/mL b – – –
culture in late exponential growth (ca 108 ± CFU/mL) on
c – – – powdered lactose monohydrate for 72 h at 41.5 ± 1°C. After
d – – – drying, total count and uninjured count per gram lactose was
e – – – estimated by plate count on Nutrient Agar and XLD agar.
a Based on the total count, a dilution of the lactose spikes
T = Test.
was performed by mixing this with a subsample of selected
matrix (for nonfat dried milk, ground black pepper) or dried
yeast extract (for dried dog food) to obtain a spiking level of
Strains from Merck Chemicals In-House Culture Collection approximately 2 CFU/g. These subsamples were left to
were not used in this part of the study. equilibrate at room temperature (20 ± 1°C) for 1 week while
(6.4.1) Methodology. the spiking level was confirmed by MPN.
(6.4.1.1) Nonselective BPW was inoculated with all After equilibration, 1.5 kg of each matrix product was
strains from cryobead/liquid stocks, and enriched for 18 ± 2 h inoculated with sufficient dried spike (about 2%) to obtain a
at 37 ± 1°C. target CFU of about 1.0–1.2 CFU/25 g and mixed by tumbling
(6.4.1.2) All enrichments were serially diluted in 0.9% for at least 30 min in a 5 kg plastic drum. The final mix was left
saline, and plate counts on Nutrient Agar were set up. Strains to equilibrate for a further 3 days (minimum) at room
reached a range of cell concentrations, from 106 to temperature prior to commencement of the analysis.
109 CFU/mL. (6.5.1.1.2) Spiking of cooked, peeled, frozen
(6.4.1.3) Portions (2–3 mL) of all undiluted BPW prawns.—Prawns for spiking were thawed overnight at +4°C.
enrichments were boiled for 15 min and allowed to cool to The drip was collected and approximately half was discarded.
room temperature (18–26°C). The remainder was spiked with a freshly grown culture of
Salmonella. This spike was added to the drained prawns and
(6.4.1.4) A 160 mL volume of boiled undiluted sample was
mixed by tumbling in a 1 L plastic bottle for 30 min at +4°C.
applied into the sample port of each Singlepath Salmonella
After mixing, the prawns were transferred to a large polythene
device.
bag and spread out to form a single layer and transferred to a
(6.4.1.5) Any presumptive cross-reacting strains were –20°C freezer. After 3 days, a subsample was removed and the
recultured into the selective RVS broth and retested undiluted. spiking level estimated by MPN. After a total of 7 days of
(6.4.2) Results.—All results obtained with the 58 strains storage, the spiked prawns were mixed with unspiked prawns
of non-Salmonella spp. are summarized in Table 6. Positive (to obtain a final spiking level of approximately 1 CFU/25 g)
signals were achieved at the control zone for all tests, by tumbling in a 5 L plastic drum for 30 min at +4°C before
indicating that all tests worked correctly. replacing at –20°C.
(6.4.2.1) Four strains cross-reacted when initially tested in The following strains from the Merck Newport In-House
the nonselective broth, BPW. When subsequently inoculated Culture Collection were used for spiking of products: nonfat
424 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

Table 5. Singlepath® Salmonella inclusivity evaluation

®
Singlepath
Salmonella result
a b
No. Source Genus Serotype Lowest CFU/mL detectable T C Serogroup

1 NCTC 8297 Salmonella Arizonae 2.20E+07 + + 51

2 MCL, Newport (SAM 0201) Salmonella Uccle 1.38E+07 + + 54
3 Robert-Koch Inst. (SAM 0641) Salmonella Kiel 1.67E+08 + + A
4 ATCC 14028 Salmonella Typhimurium 1.00E+07 + + B
5 ATCC 9842 Salmonella Abortus-equi 1.00E+07 + + B
6 ATCC 10728 Salmonella Bredeney 1.00E+07 + + B
7 ATCC 6960 Salmonella Derby 5.00E+06 + + B
8 ATCC 6961 Salmonella Essen 3.00E+07 + + B
9 ATCC 8326 Salmonella Heidelberg 2.00E+06 + + B
10 ATCC 11997 Salmonella Chester 4.00E+07 + + B
11 ATCC 6994 Salmonella Typhimurium 3.00E+06 + + B
12 ATCC 23199 Salmonella Sandiego 3.00E+06 + + B
13 MCL, Newport (SAM 0019) Salmonella Abony 8.29E+05 + + B
14 MCL, Newport (SAM 0102) Salmonella Shubra 1.65E+08 + + B
15 Robert -Koch Inst. (SAM 0642) Salmonella II Caledon 1.42E+04 + + B
16 MCL, Newport (SAM 0552) Salmonella Kubacha 8.64E+05 + + B
17 MCL, Newport (SAM 0351) Salmonella Agona 6.26E+05 + + B
18 MCL, Newport (SAM 0412) Salmonella Gloucester 2.01E+06 + + B
19 MCL, Newport (SAM 0131) Salmonella Schleissheim 1.48E+06 + + B
20 MCL, Newport (SAM 0545) Salmonella Saint-paul 3.87E+08 + + B
21 MCL, Newport (SAM 0493) Salmonella California 1.76E+06 + + B
22 Robert -Koch Inst. (SAM 0643) Salmonella San-juan 7.41E+06 + + C1
23 ATCC 51741 Salmonella Infantis 5.00E+07 + + C1
24 ATCC 13312 Salmonella Choleraesuis 3.00E+07 + + C1
25 NCTC 5742 Salmonella Virchow 3.00E+07 + + C1
26 ATCC 10722 Salmonella Tennessee 8.00E+07 + + C1
27 ATCC 8321 Salmonella Typhisuis 1.00E+07 + + C1
28 MCL, Newport (SAM 0213) Salmonella Singapore 5.59E+05 + + C1
29 MCL, Newport (SAM 0275) Salmonella Ohio 8.13E+04 + + C1
30 Robert -Koch Inst. (SAM 0644) Salmonella II Calvinia 3.74E+06 + + C1
31 Robert-Koch Inst. (SAM 0646) Salmonella Argentina 5.41E+07 + + C1
32 MCL, Newport (SAM 0005) Salmonella Lille 1.25E+08 + + C1
33 MCL, Newport (SAM 0440) Salmonella Mbandaka 1.88E+08 + + C1
34 MCL, Newport (SAM 0640) Salmonella Montevideo 1.59E+07 + + C1
35 MCL, Newport (SAM 0367) Salmonella Birkenhead 1.35E+08 + + C1
36 MCL, Newport (SAM 0441) Salmonella Mikawasima 2.69E+06 + + C1
37 MCL, Newport (SAM 0160) Salmonella Edinburgh 6.33E+06 + + C1
38 MCL, Newport (SAM 0067) Salmonella Braenderup 9.50E+06 + + C1
39 ATCC 15782 Salmonella Breukelen 6.00E+07 + + C2
40 ATCC 8388 Salmonella München 4.00E+07 + + C2
41 ATCC 12002 Salmonella Tallahassee 1.00E+06 + + C2
42 Robert-Koch Inst. (SAM 0647) Salmonella Curacao 1.59E+07 + + C2
43 ATCC 9263 Salmonella Kentucky 2.00E+08 + + C3
44 ATCC 13076 Salmonella Enteritidis 2.00E+06 + + D1
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 425

Table 5. (continued)
®
Singlepath
Salmonella result
a b
No. Source Genus Serotype Lowest CFU/mL detectable T C Serogroup

45 ATCC 10721 Salmonella Javiana 1.00E+06 + + D1

46 ATCC 15480 Salmonella Dublin 3.00E+06 + + D1
47 ATCC 9184 Salmonella Gallinarum 1.00E+07 + + D1
48 ATCC 4931 Salmonella Enteritidis 4.00E+06 + + D1
49 ATCC 19945 Salmonella Pullorum 2.00E+07 + + D1
50 ATCC 7378 Salmonella Panama 2.00E+07 + + D1
51 NCTC 0618 Salmonella Enteritidis var Dansyz 4.07E+06 + + D1
52 Robert-Koch Inst. (SAM 0649) Salmonella Mjimwema 1.73E+04 + + D1
53 NRC, Hamburg (SAM 0763) Salmonella Moscow 1.22E+05 + + D1
54 NRC, Hamburg (SAM 0764) Salmonella Blegdam 6.61E+05 + + D1
55 Robert-Koch Inst. (SAM 0718) Salmonella Portland 9.04E+04 + + D1
56 Robert-Koch Inst. (SAM 0716) Salmonella Berta 2.08E+07 + + D1
57 Robert-Koch Inst. (SAM 0717) Salmonella Victoria 8.13E+03 + + D1
58 MCL, Newport (SAM 0371) Salmonella Goettingen 8.21E+05 + + D1
59 MCL, Newport (SAM 0540) Salmonella Eastbourne 6.60E+05 + + D1
60 MCL, Newport (SAM 0050) Salmonella Rostock 2.54E+08 + + D1
61 ATCC 15793 Salmonella Maarssen 8.00E+06 + + D2
62 ATCC 9270 Salmonella Anatum 2.00E+08 + + E1
63 Robert -Koch Inst. (SAM 0653) Salmonella Oxford 1.43E+06 + + E1
64 Robert-Koch Inst. (SAM 0654) Salmonella Matroosfontein 5.42E+07 + + E1
65 MCL, Newport (SAM 0401) Salmonella Give 9.19E+07 + + E1
66 Robert-Koch Inst. (SAM 0720) Salmonella Vejle 2.84E+07 + + E2
67 ATCC 11646 Salmonella Illinois 1.00E+08 + + E3
68 Robert-Koch Inst. (SAM 0655) Salmonella Gwoza 3.29E+07 + + E4
69 NRC, Hamburg (SAM 0766) Salmonella Cannstatt 1.21E+08 + + E4
70 NRC, Hamburg (SAM 0767) Salmonella Westerstede 7.01E+06 + + E4
71 MCL, Newport (SAM 0409) Salmonella Llandoff 1.35E+08 + + E4
72 NRC, Hamburg (SAM 0770) Salmonella Yehuda 4.98E+07 + + F
73 Robert-Koch Inst. (SAM 0657) Salmonella VI unnamed 1.33E+08 + + F
74 NRC, Hamburg (SAM 0773) Salmonella II unnamed 1.70E+08 + + F
75 NRC, Hamburg (SAM 0774) Salmonella Missouri 2.77E+08 + + F
76 Robert-Koch Inst. (SAM 0728) Salmonella Friedenau 2.22E+08 + + G1
77 MCL, Newport (SAM 0062) Salmonella Kedougou 1.04E+08 + + G2
78 NRC, Hamburg (SAM 0779) Salmonella II Luanshya 6.72E+07 + + G2
79 MCL, Newport (SAM 0292) Salmonella Carrau 1.45E+08 + + H
80 NRC, Hamburg (SAM 0781) Salmonella Warragul 7.20E+07 + + H
81 ATCC 35641 Salmonella Saphra 2.00E+07 + + I
82 ATCC 8822 Salmonella Kirkee 5.00E+07 + + J
83 MCL, Newport (SAM 0003) Salmonella Cerro 1.36E+08 + + K
84 Robert-Koch Inst. (SAM 0670) Salmonella Fluntern 1.97E+08 + + K
85 Robert-Koch Inst. (SAM 0672) Salmonella Assen 2.06E+08 + + L
86 NRC, Hamburg (SAM 0794) Salmonella Aderike 2.99E+07 + + M
87 Robert-Koch Inst. (SAM 0677) Salmonella Zehlendorf 7.44E+07 + + N
88 Robert-Koch Inst. (SAM 0738) Salmonella Morningside 1.84E+08 + + N
426 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

Table 5. (continued)
®
Singlepath
Salmonella result
a b
No. Source Genus Serotype Lowest CFU/mL detectable T C Serogroup

89 Robert-Koch Inst. (SAM 0740) Salmonella II unnamed 1.80E+08 + + N

90 NCTC 7342 (SAM 0606) Salmonella Arizonae III 4.60E+07 + + O
91 NCTC 7389 (SAM 0609) Salmonella Monschaui 3.75E+07 + + O
92 Robert-Koch Inst. (SAM 0680) Salmonella Sheffield 9.52E+07 + + P
93 Robert-Koch Inst. (SAM 0683) Salmonella Wandsworth 3.70E+07 + + Q
94 MCL, Newport (SAM 0567) Salmonella IV unnamed 5.55E+07 + + R
95 Robert-Koch Inst. (SAM 0743) Salmonella Waycross 1.29E+08 + + S
96 NRC, Hamburg (SAM 0800) Salmonella Burundi 2.27E+08 + + S
97 Robert-Koch Inst. (SAM 0744) Salmonella III unnamed 9.75E+07 + + T
98 NRC, Hamburg (SAM 0805) Salmonella Irigney 1.57E+08 + + U
99 MCL, Newport (SAM 0286) Salmonella Berkeley 1.80E+08 + + U
100 MCL, Newport (SAM 0279) Salmonella Lohbruegge 2.53E+06 + + V
101 MCL, Newport (SAM 0297) Salmonella Deversoir 8.37E+07 + + W
102 MCL, Newport (SAM 0284) Salmonella Kaolack 1.32E+06 + + X
103 NRC, Hamburg (SAM 0813) Salmonella II Ngozi 1.85E+08 + + Y
104 MCL, Newport (SAM 0190) Salmonella II Greenside 5.47E+07 + + Z
105 NRC, Hamburg (SAM 0816) Salmonella IV Bonaire 8.02E+06 + + Z

a
T = Test.
b
C = Control.

dried milk: S. Newport—previously isolated from milk
powder; ground black pepper; S. Infantis—in-house isolate
from ground black pepper; dried dog food; S. Derby—in-house
isolate from Hide Sticks (“dog chews”); and cooked prawns;
S. Butantan—previously isolated from frozen prawns.
On the first day of analysis, the level of background flora
was estimated by standard plate count of dilutions series of
each type of sample.
Ten grams of sample + 90 mL maximum recovery diluent
(MRD) was stomached for 2 min. Serial dilutions were
prepared in MRD, and 0.1 mL aliquots surface were plated
onto Nutrient Agar. After incubation for 24 h at 37°C,
colonies were counted on plates showing 30–300 colonies and
the weighted mean (CFU/g) was calculated.
(6.5.1.2) Enrichment of food samples.—Immediately
prior to sampling, each food product was mixed by tumbling
in a 5 kg plastic drum for 30 min at room temperature for the
dried products and +4°C for frozen prawns.
The following samples were analyzed in each series:
(6.5.1.2.1) Test samples (25 g each).—Including
20 spiked samples and 5 unspiked samples (negative
controls). All test samples were added to 225 mL BPW,
according to the ISO 6579:2002 protocol (1). For naturally
contaminated samples of desiccated coconut, 25 samples of
25 g each were tested.
(6.5.1.2.2) MPN testing.—On each sample product, a
3 tube, 4-dilution MPN count was performed to estimate the
contamination level. For MPN determination, three 100 g,
three 10 g, and three 1 g samples were added to 900, 90, and
9 mL BPW, respectively. For the three 0.1 g MPN samples, it
was considered too inaccurate to weigh this amount. Instead,
10 g of the spiked product was added to 90 mL BPW, mixed,
and 1 mL of this suspension was added to 9 mL BPW. Samples
were allowed to wet or thaw, respectively, prior to mixing for
1 min in a Stomacher.
The ISO 6579:2002 protocol (1) was followed for sample
enrichment. All samples were diluted 1:10 in BPW and
incubated at 37 ± 1°C for 18 ± 2 h. At the end of the
pre-enrichment period, each pre-enrichment culture was
inoculated into selective enrichment broths.
Universal containers containing 10 mL RVS were
inoculated with 0.1 mL pre-enrichment culture and incubated
for 24 ± 3 h at 41.5 ± 1°C.
Universal containers containing 10 mL MKTTn
supplemented with iodine/potassium iodide solution were
inoculated with 1 mL pre-enrichment culture and incubated
for 24 ± 3 h at 37 ± 1°C.
After selective enrichment, each RVS enrichment was
analyzed by both the ISO 6579:2002 protocol (1) and the
Singlepath Salmonella protocol. All MKTTn enrichments
were analyzed by the ISO 6579:2002 protocol (1) only.
(6.5.1.3) Merck Singlepath Salmonella testing.—Testing
of RVS broth selective enrichments was performed according
to the manufacturer’s instructions. Briefly, 1–2 mL RVS broth
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 427

Table 6. Singlepathâ Salmonella exclusivity evaluation

Recultured cross-reacting

BPW RVS
Singlepath Singlepath
a d
BPW b c
RVS
No. Source Genus Species CFU/mL T C CFU/mL T C

1 ATCC 7966 Aeromonas hydrophila 2.00E+08 – +

2 ATCC 19433 Enterococcus faecalis 1.00E+09 – +
3 ATCC 11778 Bacillus cereus 1.00E+08 – +
4 ATCC 6750 Citrobacter brakii 5.30E+08 ± + 1.00E+4 – +
5 ATCC 8090 Citrobacter freundii 8.00E+08 – +
6 ATCC 27028 Citrobacter koseri 6.00E+08 – +
7 ATCC 6057 Enterococcus faecium 1.30E+09 – +
8 ATCC 11775 Escherichia coli 4.00E+09 – +
9 ATCC 29926 Hafnia alvei 2.00E+09 ± + 2.00E+07 – +
10 ATCC 13047 Enterobacter cloacae 8.00E+09 – +
11 ATCC 29544 Enterobacter sakazakii 1.70E+09 – +
12 ATCC 13048 Enterobacter aerogenes 7.70E+09 – +
13 ATCC 12228 Staphylococcus aureus 3.00E+08 – +
14 ATCC 9610 Yersinia enterocolitica 2.10E+09 – +
15 ATCC 29909 Yersinia intermedia 1.10E+09 – +
16 ATCC 6538 Staphylococcus aureus 1.20E+09 ± + 5.00E+03 – +
17 ATCC 11060 Shigella sonnei 4.00E+09 + + 1.00E+03 – +
18 ATCC 12025 Shigella flexneri 3.40E+09 + + 3.00E+05 – +
19 30124 DSMZ Serratia rubideae 8.00E+08 – +
20 ATCC 7462 Serratia plymuthica 2.50E+08 – +
21 ATCC 14756 Serratia marcescens 1.80E+09 ± + 0 – +
22 ATCC 13315 Proteus vulgaris 6.00E+09 + + 0 – +
23 ATCC 14153 Proteus mirabilis 1.00E+09 – +
24 ATCC 10240 Micrococcus luteus 1.00E+08 – +
25 ATCC 13883 Klebsiella pneumoniae 1.70E+09 – +
26 ATCC 13182 Klebsiella oxytoca 5.00E+09 – +
27 ATCC 13525 Pseudomonas fluorescens 5.00E+08 – +
28 ATCC 25830 Morganella morganii 3.00E+09 – +
29 ATCC 19209 Alcaligenes faecalis 2.00E+09 – +
30 ATCC 25015 Francicella philomiragia 1.00E+08 – +
31 ATCC 33852 Ewingella americana 3.00E+09 – +
32 ATCC 51642 Citrobacter spp 2.50E+09 – +
33 ATCC 14869 Lactobacillus brevis 2.00E+09 – +
34 ATCC 33320 Butiauxella agratis 3.50E+09 – +
35 ATCC 12633 Pseudomonas putida 1.60E+08 – +
36 ATCC 11509 Brochotrix thermosphacta 5.00E+08 – +
37 ATCC 33855 Cedecea neteri 1.00E+09 – +
38 ATCC 23724 Escherichia coli C600 7.00E+08 – +
39 ATCC 19615 Streptococcus pyogenes <1.00E+07 – +
428 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

Table 6. (continued)
Recultured cross-reacting

BPW RVS
Singlepath Singlepath
a d
BPW b c
RVS
No. Source Genus Species CFU/mL T C CFU/mL T C

40 ATCC 23746 Escherichia coli 1.70E+09 – +

41 ATCC 13159 Providencia rustigianii 1.00E+09 – +
42 ATCC 7644 Listeria monocytogenes 6.00E+08 – +
43 ATCC 12759 Bacillus licheniformis 1.50E+6 – +
44 ATCC 19606 Acinetobacter baumannii 1.00E+08 – +
45 ATCC 25629 Rhodocuccus equi 1.70E+08 – +
46 ATCC 13722 Corynebacterium bovis 1.00E+09 – +
47 ATCC 19120 Listeria grayi 5.00E+08 – +
48 ATCC 10231 Candida albicans <1.00E+07 – +
49 ATCC 33433 Kluyvera ascorbata 1.00E+09 + + 0 – +
50 ATCC 29473 Yersinia ruckeri 1.00E+09 – +
51 ATCC 17802 Vibrio parahaemolyticus 4.00E+08 – +
52 ATCC 35236 Yersinia aldovae 1.50E+09 – +
53 ATCC 8702 Shigella boydii 6.00E+09 – +
54 ATCC 29844 Serratia fonticola 2.00E+09 – +
55 ATCC29911 Yersinia kristensenii 5.00E+09 – +
56 ATCC 33641 Yersinia frederiksenii 5.00E+09 – +
57 ATCC 29833 Yersinia pseudotuberculosis 1.00E+09 – +
58 ATCC 9763 Saccharomyces cerevisiae 2.00E+06 + + 0 – +

a
BPW = Buffered peptone water.
b
T = Test.
c
C = Control.
d
RVS = Rappaport-Vassiliadis soy broth.

enrichment was transferred to a 3 mL polypropylene tube with
a disposable Pasteur pipet. The tubes were placed in an
appropriate rack and loosely capped. The rack was placed in a
water bath with boiling water, the bath was covered with a lid,
and samples were heat treated for 15 min. After heat
treatment, the rack was transferred to room temperature and
the boiled samples were left to cool.
After cooling, 160 ± 3 mL boiled sample was pipetted into
the sample well of a Singlepath device. The tests were read
after 20 min. All samples showing a pink line (however faint)
at the test-line position as well as at the control-line position
were scored as positive. Devices showing only a pink line at
the control-line position were scored as negative. As the RVS
enrichments tested were subsampled from the ISO 6579:2002
cultures, reference method (1), confirmation of all results was
obtained from the results of the ISO 6579:2002 reference
method (1).
(6.5.1.4) ISO 6579:2002 isolation and confirmation
method (1).—Each selective enrichment (RVS broth and
MKTTn broth) was streaked with a sterile, disposable 10 mL
loop onto XLD and HE agars. The plates were incubated at
37 ± 1°C for 24 ± 3 h. At least 1 suspect colony from each type
of selective enrichment from each sample was picked and
streaked onto Nutrient Agar. Pure cultures were tested by
Poly O and Poly H sero-agglutination and biochemically
identified using API 20 E.
(6.5.1.5) Results.
(6.5.1.5.1) Results are summarized in Tables 7 and 8.
Fractional positives were achieved at the low spiking level of
0–1.08 CFU/25 g for all foods tested.
(6.5.1.5.2) All enrichments presumed Salmonella spp.
positive by Singlepath were confirmed positive by the ISO
reference method, including serotyping, and the BioMerieux
API identification system.
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 429

Uninjured, %

4.52
2.47
0.92
Post storage of spike at room temperature
Table 7. Summary of total and uninjured counts used for determining inoculation level of the singular lots of food matrixes in the internal validation study

XLD Agar,

3
Uninjured

3.14 ´ 10
7.15 ´ 10
3.40 ´ 10
CFU/g
count

Nutrient Agar,
Total count

5
6.95 ´ 10
2.89 ´ 10
3.68 ´ 10
CFU/g Uninjured, %

6.93
0.62
NT
Post 48 h drying of spike on lactose

Uninjured count

1.05 ´ 103
3
XLD Agar,

7.00 ´ 10
CFU/g

b
NT
Figure 2. Flow diagram of methodology for food
testing. Nutrient Agar,

(6.5.1.5.3) All remaining spiked and unspiked (control)

Total count

5
1.02 ´ 10
1.01 ´ 10
1.70 ´ 10
CFU/g

enrichments were confirmed Salmonella spp. negative.

7 Independent Validation Studies

Validation studies were conducted by r-tech laboratories

XLD Agar,

under the direction of the AOAC Research Institute.

a
CFU/g
background count

0
0
0
0
0
0
Food matrix

Total count

7.1 Method Comparison Study

(7.1.1) Methodology.—Raw ground beef and raw ground
Agar, CFU/g

2
4.27 ´ 10
Nutrient

4.5 ´ 10
3.3 ´ 10

turkey were collected fresh from the local supplier.

0
0
0

(7.1.1.1) The 2 foods were screened before inoculation

with the test organism, for indigenous bacteria/Salmonella.
(7.1.1.2) A singularly inoculated lot of each food was then
XLD = Xylose lysine desoxycholate.
37°C Cooked, peeled, frozen prawns
30°C Cooked, peeled, frozen prawns

prepared. The low and high target spike levels were the same
for both foods: a low target spike level of 0.6 CFU/25 g, and a
high target spike level of 1 CFU/25 g.
Dried skimmed milk powder

(7.1.1.3) S. Typhimurium, ATCC 14028, was used to

inoculate raw ground beef, and S. Enteritidis, ATCC 13076,
Desiccated coconut

was used to inoculate raw ground turkey.

NT = Not tested.

(7.1.1.4) The ISO 6579:2002 protocol (1) was followed

Dried dog food
Black pepper

for blending/mixing and the enrichment procedure. Because

Food matrix

the sample enrichment schemes for the reference method and

the Singlepath method were the same, forty-five 25 g samples
were tested: 20 samples with a low target spike level of
a
b
430 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

Specificity False negative False positive Chi square

0.6 CFU/25 g, 20 samples with a high target spike of

value

1.0

1.0
1.0

1.0

1.0
1 CFU/25 g, and 5 uninoculated samples which served as
negative controls. Samples for MPN determination were
processed with the test portions.
(7.1.1.5) Enrichment.
rate, %

(7.1.1.5.1) From each spiking level, the 25 g test portions

0
0

0
were enriched in 225 mL BPW and incubated at 37°C for
18 ± 2 h. At the end of the enrichment period, each enriched
test portion was transferred to selective enrichments RVS
rate, %

broth and MKTTn broth supplemented with iodine/potassium

0

0
0

0
iodide solution. The enrichments were incubated for 24 ± 3 h
at 41.5 and 37°C, respectively.
(7.1.1.5.2) After selective enrichment at 24 ± 3 h at 41.5°C
rate, %

for RVS and 37°C for MKTTn, each sample was analyzed by
100

100
100

100

100
both the ISO 6579:2002 protocol (1) and the Singlepath
Salmonella protocol.
(7.1.1.6) Singlepath Salmonella analysis.
Sensitivity
rate, %

(7.1.1.6.1) The Singlepath protocol is designed for RVS

100

100
100

100

100
Table 8. Summary of Singlepath® Salmonella and ISO reference method results for the internal validation study

enrichments only; therefore, the MKTTn enrichments were

not tested by this method.
(7.1.1.6.2) It was not required to test MPN enrichments by
positive/total food

this method.
portions tested
ISO reference
confirmed

(7.1.1.6.3) Boiled samples were prepared by adding 1 mL

16/20

23/25
10/20

13/20

11/20
0/5

0/5

0/5

0/5

of each sample to a polypropylene tube and boiled for 15 min

in water.
(7.1.1.6.4) Boiled samples were allowed to cool to room
temperature before testing.
positive/total food positive/total food
portions tested

(7.1.1.6.5) A 160 mL volume of boiled sample was applied

®

confirmed
Singlepath

to the sample port of a Singlepath device.

16/20

23/25
13/20
10/20

11/20
0/5

0/5

0/5

0/5

(7.1.1.6.6) After 20 min at room temperature, the results

were observed and recorded.
(7.1.1.7) ISO 6579:2002 reference method analysis (1).
(7.1.1.7.1) Using a 10 mL loop, all RVS and MKTTn
portions tested
presumptive
®

enrichments, including MPN enrichments, were streaked onto

Singlepath

16/20
13/20

23/25
10/20

11/20
0/5

0/5

0/5

0/5

XLD and bismuth sulfite agar. The XLD agar was incubated at
37°C for 24 ± 3 h and the bismuth sulfite agar was incubated at
35°C for 48 ± 2 h.
(7.1.1.7.2) After incubation, a colony exhibiting typical
Contamination
level/sample,
a

Salmonella morphology on each medium was confirmed by

CFU/25 g

0.58
1.08

1.08
1.08
0.23

streaking onto a Nutrient Agar plate, incubating for 24 ± 3 h at

0

37°C, and then testing by the biochemical identification Vitek

As determined by 3-tube, 4-dilution MPN procedure.

system, using a GNI+ card.

Desiccated coconut, naturally contaminated sample

(7.1.1.7.3) Isolates identified as Salmonella species by

Vitek were then confirmed by serology using Poly O and
Cooked, peeled, frozen prawns, control sample
Cooked, peeled, frozen prawns, test sample

Poly H antisera.
Dried skimmed milk powder, control sample
Dried skimmed milk powder, test sample

(7.1.2) Results.
(7.1.2.1) Results are summarized in Table 9.
(7.1.2.2) The Merck Singlepath Salmonella Gold Labeled
Dried dog food, control sample

ImmunoSorbent Assay (GLISA) can yield a presumptive

Black pepper, control sample
Dried dog food, test sample

result for the presence of Salmonella spp. in raw ground beef

Black pepper, test sample

and raw ground turkey 48 h faster than the ISO reference

method (1).
(7.1.2.3) When testing raw ground beef, there were no
false-negative or false-positive results.
Food matrix

(7.1.2.4) In the case of raw ground turkey, there were

false-positive results (3 low inoculation level, 4 high
inoculation level). In all 7 cases, no less than 5 different
a
 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006 431

False-positive Chi square

isolates were tested. In 6 of the 7 false-positive results, a

value

1.0
1.0

0.8
0.0
swarming Proteus was the organism recovered. In one case, a
Citrobacter was the organism recovered.
(7.1.2.5) When testing raw ground turkey, there were a
total of 3 false-negative results (2 low inoculation level, 1 high
rate, %

33.3
inoculation level).

15
0
0
(7.1.2.6) Results were clearly and easily interpreted.
8 Discussion
False-negative

8.1 Ruggedness
rate, %

7.7
40
0
0

(8.1.1) There was a negative result for unboiled

S. Typhimurium on Day 2. This could be due, first, to the use
Table 9. Summary of Singlepath® Salmonella and ISO reference method results for the independent method comparison study

of fresh cultures on Day 2. Antigen expression may have

differed for Day 1 cultures. Second, the antibody used in
Specificity
rate, %

Singlepath Salmonella has been raised against heat-killed

100
100

67
85

cells. Boiled cultures therefore tend to give a better

performance than unboiled. The boiling time study showed
that completely omitting the sample boiling step could
Sensitivity
rate, %

compromise the results.

100
100

92
60

(8.1.2) Leaving the samples boiling for 10 min longer than

the recommended 15 min stated in the product insert did not
have a significant adverse effect on the test performance.
confirmed positive/total
food portions tested

(8.1.3) The sample result observation time study showed

ISO Reference

that reading results 5 min earlier or later than specified in the

14/20
11/20

13/20

product insert did not have a significant adverse effect on the

5/20
0/5

0/5

test performance.
(8.1.4) The assay sample volume study showed that
varying the sample volume recommended in the product
insert ±10 mL did not have a significant adverse effect on the
positive/total food

test performance.
portions tested
®

confirmed
Singlepath

14/20
11/20

16/20
6/20

8.2 Lot-to-Lot Testing

0/5

0/5

(8.2.1) This study showed that Singlepath Salmonella

gave a consistent performance between the 3 lots of test kits
analyzed.
positive/total food
portions tested

8.3 Inclusivity Study

presumptive
®
Singlepath

14/20
11/20

16/20
6/20
0/5

0/5

(8.3.1) This study showed that Singlepath Salmonella

detected many different serovars of Salmonella.
(8.3.2) The detection limit varied with serogroup from
104 to 108 CFU/mL.
As determined by 3-tube, 4-dilution MPN procedure.
Contamination

(8.3.3) The strength of the test signal varied with

level/sample,
a
CFU/25 g

serogroup.
<0.075

<0.075
1.075

2.325
0.525
6

8.4 Exclusivity Study

(8.4.1) This study showed that Singlepath Salmonella
discriminated Salmonella spp. from organisms belonging to
Control

Control
Spike

High

High
level

Low

Low

selected non-Salmonella genera.

(8.4.2) The strains that produced positive signals in
nonselective BPW were recultured in the selective RVS broth,
and when retested produced no cross-reactions with Singlepath
Raw ground turkey
Raw ground beef

Salmonella. The selective broth prevented the growth and

false-positive reactions of these organisms.
(8.4.3) The 5 strains that cross-reacted in BPW and were
culturable in RVS, but negative when retested by Singlepath
Food

Salmonella might not have grown to the cell density that

a
432 THOMPSON & LINDHARDT: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 2, 2006

Table 10. Summary of internal and independent (8.6.1.4) The Merck Singlepath Salmonella GLISA
method comparison study results
yielded a presumptive result for the presence of Salmonella
®
Singlepath Salmonella spp. in raw ground beef and raw ground turkey 24 h faster
ISO 6579:2002 Positive Negative Total
than the ISO reference method (1).
8.7 Internal and Independent Method Comparison
Study
Positive 113 3 116
Negative 7 92 99 (8.7.1) Results are summarized in Tables 10 and 11.
Total 120 95 215 (8.7.2) With reference to Table 11, overall, the Chi-square
value of 0.9 indicated that there was no significant difference
between Singlepath Salmonella and the ISO 6579:2002
reference method (1).
would cause a problem in the selective broth. These strains
(8.7.3) Overall, Singlepath Salmonella gave a low
are, therefore, unlikely to cause false-positive results.
false-positive rate of 7.3% and a low false-negative rate of 2.5%.
8.5 Internal Method Comparison Study (8.7.4) It is possible that the specificity rate should be
higher than 93%, as the swarming Proteus spp. in the raw
(8.5.1) This study showed 100% agreement between the
ground turkey may have masked the presence of the target
Singlepath Salmonella method and the ISO 6579:2002
Salmonella spp.
reference method (1).
(8.5.2) The Singlepath method did not produce any
9 Conclusions
false-positive or false-negative results.
(8.5.3) The Singlepath method yielded a presumptive result
The internal and independent method comparison
for the presence of Salmonella spp. in the food matrixes tested
evaluations of the Singlepath Salmonella assay clearly
up to 24 h faster than the ISO 6579:2002 reference method (1).
demonstrated that this method is equivalent to the
8.6 Independent Method Comparison Study ISO 6579:2002 reference method (1) for detection of
Salmonella spp. at spiking levels ranging from low
(8.6.1.1) This study showed 100% agreement between the
(0.23–1.08 CFU/25 g) to high (2.3–6.0 CFU/25 g) in the
Singlepath Salmonella method and the ISO 6579:2002
following selected foods: dried skimmed milk, black pepper,
reference method (1) for raw ground beef.
dried pet food, desiccated coconut, cooked peeled frozen
(8.6.1.2) When testing raw ground beef, there were no
prawns, raw ground beef, and raw ground turkey. The
false-negative or false-positive results.
inclusivity and exclusivity data demonstrated that Singlepath
(8.6.1.3) When testing raw ground turkey, there were
Salmonella can detect most serogroups of Salmonella and can
3 false positives from 20 test samples at the low inoculation
discriminate Salmonella spp. from organisms belonging to
level, and 4 false positives from 20 test samples at the high
selected non-Salmonella genera. The lot-to-lot, stability, and
inoculation level. In 6 of the 7 false-positive results, a
ruggedness data also presented evidence that Singlepath
swarming Proteus was the organism recovered. In one case, a
Salmonella is a stable and robust assay and can be consistently
Citrobacter was the organism recovered. It is possible that the
manufactured in reproducible quality. Singlepath Salmonella
target organism may have been present at low titer, but
can be recommended as a rapid presumptive qualitative assay
masked by the high background flora and, therefore, not
for the detection of Salmonella spp. in foods.
recoverable by standard methods.
10 References
Table 11. Internal and independent method
comparison study performance indicators and the test (1) ISO 6579:2002 (4th Ed.), Microbiology of Food and Animal
for significant differencea Feeding Stuffs–Horizontal Method for the Detection of
Salmonella spp., International Organization for
Indicator Value
Standardization, Geneva, Switzerland
(2) Microbiology Laboratory Guidebook (2003) Chapter 4,
Sensitivity rate, % 97 Revision 02: Isolation and Identification of Salmonella from
Meat, Poultry and Egg Products, USDA–FSIS, Washington,
Specificity rate, % 93
DC, http://www.fsis.usda.gov/PDF/MLG_4_03.pdf
False-negative rate, % 2.59 (3) Bacteriological Analytical Manual Online (April 2003)
False-positive rate, % 7.07 Chapter 5, Salmonella, U.S. Food and Drug Administration,
Chi-square 0.9 Center for Food Safety and Applied Nutrition, College Park,
MD, http://www.cfsan.fda.gov/~ebam/bam-5.html
a
Determined using formulae outlined in AOAC Microbiological
®
Guidelines for Methods Validation, Appendix 4, Singlepath
Salmonella Validation Outline v4.