3M Food Safety

Technical Bulletin
Number: TB.171049.02 Technology Platform: Pathogens
Effective Date: Feb. 15, 2012 Originating Location: St. Paul, MN
Supersedes: TB.171049.01

3M™ Molecular Detection Assay Salmonella

Performance Summary
Salmonella is one of the most common causes of food-borne illnesses and causes an estimated 2 to 4 million cases of
salmonellosis in the US annually1. A comprehensive farm-to-table approach to food safety is needed to reduce salmonellosis.
Faster and more accurate testing methods are required versus the traditional microbiology methods which are cumbersome
and take more than four days to get results. The 3M™ Molecular Detection Assay Salmonella was developed for the rapid
and specific detection of Salmonella spp. in food and food process environmental samples after enrichment in 3M™ Buffered
Peptone Water ISO (BPW ISO).
Performance of the 3M method was evaluated for selectivity, with a range of food matrices, non-food samples and compared
to the reference methods. The limit of detection of the method was also determined.

Method Selectivity – Inclusivity and Exclusivity Testing

Inclusivity is defined as the ability of a method to detect the target analyte from a wide range of strains.
Exclusivity is defined as the lack of interference from a relevant range of non-target strains.

Pure bacterial cultures were derived from purchased lyophilized preparations or from frozen stock cultures or identified by
biochemical methods.

a. False negative rate – Inclusivity

One hundred and four (104) Salmonella isolates (Appendix 1) were tested. The Salmonella strains were cultured
overnight then diluted to a level of less than 105 CFU/mL (Colony Forming Unit/mL) prior to testing. A false-negative rate
of  1% (> 99% inclusivity) was determined. Only one strain of one serotype, Salmonella enterica Westhampton*, was
shown to be negative by the 3M method.

b. False positive rate – Exclusivity

Fifty (50) non-Salmonella isolates (Appendix 2) were tested. The non-Salmonella strains were cultured to reach a minimum
level of 100X the limit of detection prior to testing. A false-positive rate of 0 % (100% exclusivity) was determined.

*Salmonella enterica Westhampton was shown to be negative by the 3M

Inclusivity (%) > 99* method. It is widely recognized that the invA gene is a pathogenicity marker
for defining Salmonella genus. 2-7 While this strain may be identified as
Salmonella, it may lack the required pathogenicity gene and would be
Exclusivity (%) 100 considered atypical and non-pathogenic

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3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
Food Studies
Food studies were conducted to assess the performance of the method in the presence of possible interferences from sample matrix
and other organisms.

Study Design
Strains of Salmonella Typhimurium, S. Enteritidis and S. Newport were used to artificially contaminate food samples commonly
reported in outbreaks and/or reported to be challenging due to their composition, i.e. high fat, high calcium, high native microflora, etc

Sixty-seven (67) different food matrices were evaluated as follows: either spiked at a level of 7-13 CFU Salmonella per sample size
or as enriched blanks (Appendix 3). All enrichments were performed at a 1:10 dilution in pre-warmed 3M BPW ISO and were
incubated for 18 hours at 37 C. All artificially contaminated samples were tested using both the 3M Molecular Detection Assay
Salmonella and the 3M Molecular Detection Matrix Control, while enriched blanks were tested using the 3M Molecular Detection
Assay Salmonella only. All enrichments were also streaked onto BBL™ CHROMagar™ Salmonella and/ or XLD agar plates, used as
a reference point.

The method was shown to be compatible with a variety of relevant food matrices. Three samples, oregano leaves, cinnamon and a
very specific brand of orange juice from concentrate, yielded unexpected negative results due antimicrobial effects from the sample
on the target growth. Spiking of these samples post-enrichment with Salmonella yielded positive result as expected providing
confirmation that the target growth in 3M BPW ISO was inhibited by the sample matrix. In these cases, a larger dilution such as1:100
or higher would be recommended as described in the FDA-BAM Chapter 5 Salmonella 8 reference method (section ) and EN ISO
6887- 4 9 (section 9.5.4.4).

Environmental and Poultry Rinse Studies

A variety of environmental samples and poultry rinses were also tested to evaluate the performance of the method in the presence of
possible interference from matrix and/or competing microbiota.

Study Design
One hundred and forty two (142) different environmental samples, including poultry drag swabs, were collected in duplicate from
various farms. Surfaces tested included concrete floors and ceiling, wooden roosts, metal feeders, and stainless steel equipment and
pipes. Eighty-four (84) bird rinses were provided by 9 different poultry processing plants. Sample collection devices and hydrating
solutions used in this study, and enrichment volume of 3M BPW ISO added to each type of sample are summarized below.

3M BPW ISO Enrichment

Sampling Device Sample Type Hydrating Solution
Volume (mL)
None Poultry carcass rinse BPW 30 mL
3M™ Dry-Sponge Surfaces 10 mL D/E Neutralizing Broth 50 mL
3M™ Dry-Sponge
Poultry fecal drag swab 10 mL BPW 50 mL
with String

Two sample sets were tested as follows:

Set 1: Suspension of Salmonella Typhimurium, S. Enteritidis or S. Newport were used to artificially contaminate one of the
duplicates with ~10 CFU. The other duplicate was enriched blank.
Set 2: Single samples enriched as blank to evaluate for natives.
All enrichments were performed at 37 C for 18 hours then tested using the 3M Molecular Detection Assay Salmonella and the 3M
Molecular Detection Matrix Control. Enrichments were also either streaked onto chromogenic BBL™ CHROMagar™ Salmonella agar
plates or tested by q-PCR, as reference points. A Chi-square (X2) test was used to compare the results for significant differences.

TB. 171049.02 Page 2 of 11

3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)

Target Type of Sample n Accuracy % Specificity % Sensitivity % X2

Environmental 142 99 100 95 0.00

Salmonella
Bird Rinse 84 97.6 100 93.9 1.33

Accuracy, specificity and sensitivity of the 3M method were calculated according to the statistical formula in Appendix 4. No
significant differences were observed between the 3M method results and the chromogenic agar results or q-PCR results as
indicated by a X2 value of less than 3.84.The method was shown to be compatible with a variety of environmental samples, including
poultry drag swabs and bird rinses. The 3M Molecular Detection Matrix Control yielded valid results for all samples tested, indicating
no interference by the variety of environmental matrices evaluated.

Methods Comparison
A study was performed with contaminated samples to obtain fractionally positive results, those in which at least one of the methods
(candidate or reference) yields 5-15 positive results out of 20 replicates examined for the low level of inoculation, for each matrix
tested.

The 3M Molecular Detection Assay Salmonella method was compared to the US FDA-BAM Chapter 5 Salmonella reference method
for raw frozen shrimp, fresh spinach and wet pet food, and to the USDA FSIS MLG 4.05 Isolation and Identification of Salmonella
from Meat, Poultry, Pasteurized Egg, and Catfish Products10 reference method for chicken nuggets, raw ground beef, and liquid egg.
Twenty (20) replicates of each matrix were analyzed at 0.2-2 CFU per test portion. Five (5) control replicates were also analyzed at 0
CFU/test portion (un-inoculated). Inoculums were stressed to mimic food processing and spiked samples were conditioned to mimic
natural contamination.

Contamination level, MPN

Sample Reference Method Χ2
per sample size(g)
Chicken nuggets 1.1 / 325 USDA FSIS MLG 4.05 0.099
Raw ground beef 0.058 / 25 USDA FSIS MLG 4.05 1.71
Raw frozen shrimp 2.3 / 100 FDA-BAM Chapter 5 1
Liquid egg 2.3 / 25 USDA FSIS MLG 4.05 3.49
Fresh spinach 0.23 / 25 FDA-BAM Chapter 5 0.41
Pet food 0.58 / 375 FDA-BAM Chapter 5 1

Results using the 3M Molecular Detection Assay Salmonella method were found to be equal to or better than the reference method
results in relevant matrices, as indicated by a X2 value of less than 3.84.

Limit of Detection (LOD)

The limit of detection of a method is defined as the lowest concentration point where reliable analytical results can be obtained. This
may vary with different serotypes and methods. For the 3M Molecular Detection Assay Salmonella this has been demonstrated to be
1-5 CFU/ sample size.

The LOD of a qualitative assay is often determined following enrichment such that at least 1 CFU in the sample size has sufficiently
multiplied and reached a level to be detected. Pathogen detection technologies have varying inherent LOD. For example, LODs for

TB. 171049.02 Page 3 of 11

3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
immunoassays are generally 105-106 CFU/mL whereas PCR LODs can be less than or equal to 104 CFU/mL of target pathogen in
the enrichment 11, 12. LOD may also vary by food matrix, bacteria in the sample and the comparison method.

One way to measure the LOD is to perform an extensive comparison study against a reference method using multiple food matrices
and multiple organisms. The LOD will depend heavily on all variables within the study, such as: the food matrix, the bacteria in the
sample and the comparison reference method. Another way to measure LOD is to test suspensions of pure target pathogen and of
known concentrations, and then test the assay for its ability to detect the target.

3M internal studies have shown that the average LOD of the 3M Molecular Detection Assay Salmonella is approximately 8.8 x 103
CFU/mL in 3M BPW ISO.

References:

1. US FDA Bad Bug Book – online:

http://www.fda.gov/food/foodsafety/foodborneillness/foodborneillnessfoodbornepathogensnaturaltoxins/badbugbook
2. Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galán JE, Ginocchio C, Curtiss R 3rd, Gyles CL. 1992. Amplification of an invA
gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol.
Cell. Probes. 6(4): 271-279.
3. Hoofar J, Ahrens P, Rådström P. 2000. Automated 5’ nuclease PCR assay for identification of Salmonella enterica. J. Clin.
Micorbiol. 38(9): 3429-3235.
4. Collazo CM, Galán JE. 1997. The invasion-associated type-III protein secretion system in Salmonella- a review. Gene.
11;192(1): 51-59. Review.
5. Ginnochio CC, Galán JE. 1995. Functional conservation among members of the Salmonella typhimurium InvA family of proteins.
Infect. Immun. 63(2):729-732.
6. Malomy B, Hoofar J, Bunge C, and Helmuth R. 2003. Multicenter validation of the analytical accuracy of Salmonella PCR:
towards an international standard. Appl. Environ. Microbiol. 69(1):290-296.
7. Steele-Mortimer O, Brumell JH, Knodler LA, Méresse S, Lopez A, Finlay BB. 2002. The invasion-associated type III secretion
system of Salmonella enteric serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial
cells. Cell Microbiol. 2002. Jan;4(1):43-54.
8. US Food and Drug Administration Bacteriological Analysis Manual on-line 2011. Chapter 5 Salmonella.
9. EN ISO 6887-4: 2004. Microbiology of food and Animal feeding stuffs. Preparation of test samples, initial suspension and
decimal dilutions for microbiobiological examination. Part 4: Specific rules for the preparation of products other than milk and
milk products, meat and meat products, and fish and fishery products.
10. US Department of Agriculture (USDA) Microbiology Laboratory Guidebook on-line 2011. MLG 4.05. Isolation and Identification of
Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products.
11. van der Z, Huis in’t Veld JH. 1997. Rapid and Alternative Screening Methods for Microbiological Analysis. Journal of AOAC
International. 80(4): 934-940.
12. Mandal PK, Biswas AK, Choi K, Pal UK. 2011. Methods for Rapid Detection of Foodborne Pathogens: An Overview. American
Journal of Food Technology. 6 (2): 81-102.

Trademarks:

BBL is a trademark of Becton, Dickinson and Company.

CHROMagar is a trademark of Dr. A. Rambach.

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3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
Appendix 1 – Salmonella inclusivity test strains
Genus, species, subspecies Serotype/ Group ID
Salmonella enterica enterica Abaetetuba TICC 2736, IMVS 1776
Salmonella enterica enterica Aberdeen TICC 2500
Salmonella enterica enterica Abony NCTC 6017
Salmonella enterica enterica Adelaide TICC 2501
Salmonella enterica enterica Agona SGSC 2458
Salmonella enterica enterica Amsterdam TICC 2660
Salmonella enterica enterica Anatum TICC 2668
Salmonella enterica enterica Augustenborg TICC 2670
Salmonella enterica enterica Birkenhead TICC 1273
Salmonella enterica enterica Blockley ATCC 51961
Salmonella enterica enterica Bovis-morbificans TICC 1293
Salmonella enterica enterica Braenderup TICC 2508
Salmonella enterica enterica Brandenburg TICC 2674
Salmonella enterica enterica Brazil NCTC 8446
Salmonella enterica enterica Bredeney TICC 2510
Salmonella enterica enterica Cerro TICC 2176
Salmonella enterica enterica Choleraesuis ATCC 10708
Salmonella enterica enterica Derby TICC 2519
Salmonella enterica enterica Dublin TICC 2757
Salmonella enterica enterica Emek TICC 1447
Salmonella enterica enterica Enteritidis ATCC 13076, CDC K-1891
Salmonella enterica enterica Give TICC 1282
Salmonella enterica enterica Hadar TICC 2528
Salmonella enterica enterica Havana TICC 443
Salmonella enterica enterica Heidelberg TICC 561
Salmonella enterica enterica Hessarek TICC 2655
Salmonella enterica enterica Infantis TICC 2616
Salmonella enterica enterica Johannesburg TICC 2543, IMVS 8346
Salmonella enterica enterica Kedougou TICC 4102
Salmonella enterica enterica Kentucky TICC 2620, IMVS 7873
Salmonella enterica enterica Kiambu TICC 2692, IMVS 5468
Salmonella enterica enterica Kimberley TICC 778
Salmonella enterica enterica Kokomlemle TICC 2544, IMVS 8652
Salmonella enterica enterica Kottbus TICC 2621, IMVS 7768
Salmonella enterica enterica Lansing TICC 2622, IMVS 7782

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3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
Appendix 1 (cont) – Salmonella inclusivity test strains
Genus, species, subspecies Serotype/ Group ID
Salmonella enterica enterica Lexington TICC 2624, IMVS 7450
Salmonella enterica enterica Lille TICC 2684, IMVS 6854
Salmonella enterica enterica Litchfield TICC 2547, IMVS 8902
Salmonella enterica enterica Livingstone TICC 2549, IMVS 8566
Salmonella enterica enterica Llandoff TICC 1291
Salmonella enterica enterica London TICC 2550, IMVS 8897
Salmonella enterica enterica Manhattan TICC 2551
Salmonella enterica enterica Mbandaka TICC 1289
Salmonella enterica enterica Montevideo TICC 763
Salmonella enterica enterica Muenchen SGSC 2490, SARB33
Salmonella enterica enterica Naestved SGSC 3612
Salmonella enterica enterica Newport ATCC 6962
Salmonella enterica enterica Nottingham NCTC 7832
Salmonella enterica enterica Ohio TICC 444
Salmonella enterica enterica Oranienburg TICC 2560
Salmonella enterica enterica Orion TICC 720
Salmonella enterica enterica Panama SGSC 2496, SARB39
Salmonella enterica enterica Paratyphi A SGSC 2499, SARB42
Salmonella enterica enterica Paratyphi B TICC 2240
Salmonella enterica enterica Paratyphi C SGSC 3592, 4034-88
Salmonella enterica enterica Poona TICC 2472
Salmonella enterica enterica Pullorum (Gallinarum) ATCC 13036
Salmonella enterica enterica Reading SGSC 2510, CGSC 2510; L32
Salmonella enterica enterica Rissen TICC 2644
Salmonella enterica enterica Rubislaw TICC 2645
Salmonella enterica enterica Saint Paul TICC 559
Salmonella enterica enterica Schwarzengrund TICC 4513, IMVS 3094
Salmonella enterica enterica Sendai SGSC 2515, SARB58
Salmonella enterica enterica Senftenberg TICC 2178
Salmonella enterica enterica Singapore TICC 2652
Salmonella enterica enterica Stanley SGSC 2517
Salmonella enterica enterica Tennessee TICC 2577, IMVS 8493
Salmonella enterica enterica Thompson SGSC 2519, SARB62
Salmonella enterica enterica Typhi ATCC 6539
Salmonella enterica enterica Typhimurium ATCC 14028, NCTC 12023, NCIMB 13284

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3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
Appendix 1 (cont) – Salmonella inclusivity test strains
Genus, species, subspecies Serotype/ Group ID
Salmonella enterica enterica UNNAMED SGSC 3038
Salmonella enterica enterica Virchow TICC 552
Salmonella enterica enterica Virginia TICC 2659
Salmonella enterica enterica Wandsworth TICC 2661, IMVS 8107
Salmonella enterica enterica Waycross TICC 566
Salmonella enterica enterica Welikade TICC 2586, IMVS 8887
Salmonella enterica enterica Weltevreden TICC 2663, IMVS 7870
Salmonella enterica enterica Westhampton TICC 3053
Salmonella enterica enterica Wien SGSC 2528, SARB71
Salmonella enterica enterica Worthington TICC 2587, IMVS 8674
Salmonella enterica enterica Zanzibar TICC 2589, IMVS 8611
Salmonella enterica enterica Zehlendorf TICC 2395, IMVS 1707
Salmonella enterica salamae (II Bleadon) SGSC 2415, S-820
Salmonella enterica salamae (II Dar-es-Salaam) SGSC 2425, SA4406
Salmonella enterica salamae (II Phoenix) SGSC 2565, CDC696-84
Salmonella enterica salamae (II Setubal) SGSC 2567, CDC42-87
Salmonella enterica arizona III a TICC 564
Salmonella enterica arizona III a TICC 2457
Salmonella enterica arizona III a NCTC 8297
Salmonella enterica diarizonae III b ATCC 43973, NCTC 10060
Salmonella enterica diarizonae III b ATCC 29934
Salmonella enterica diarizonae III b ATCC 12325
Salmonella enterica houtenae (IV Argentina) SGSC 2555, S-1344,
Salmonella enterica houtenae (IV Wassenaar) SGSC 2576, CDC121-87
Salmonella enterica houtenae IV SGSC 3084, RKS3025
Salmonella enterica houtenae IV NCTC 10401
Salmonella bongori bongori (V Brookfield) SGSC 2579, CDC750-72
Salmonella bongori bongori (V Malawi) SGSC 2577, CDC327-80
Salmonella bongori bongori (V Maregrosso) SGSC 2578, CDC235-77
Salmonella bongori bongori V SGSC 3106, RKS3047
Salmonella enterica indica (VI Flint) SGSC 2554, S-942
Salmonella enterica indica (VI Vrindaban) SGSC 2582, CDC2083-62
Salmonella enterica indica VI SGSC 3116, RKS2995
Salmonella enterica indica VI SGSC 3120, RKS3013

Abbreviations: TICC - Tecra International Culture Collection; IMVS - Institute of Medical and Veterinary Science; SGSC - Salmonella Genetic
Stock Center; ATCC - American Type Culture Collection; NTCT- National Collection of Type Cultures; SARB - Salmonella Reference collection B;
CDC - Center for Disease Control; NCIMB – National Collection of Industrial Marine and food bacteria; others - isolates from naturally
contaminated samples

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3M™ Molecular Detection Salmonella – Performance Summary (cont.)
Appendix 2 – Non-Salmonella exclusivity test strains

Genus Species/ Serotype ID

Cedecea neteri ATCC 33855
Citrobacter brakii ATCC 29063
Citrobacter diversus TICC 1604
Citrobacter freundii ATCC 8090
Citrobacter koseri TICC 4469
Citrobacter sedlakii TICC 4088
Citrobacter youngae TICC 4255
Cronobacter sakazakii ATCC 29544
Edwardsiella tarda NCTC 10396
Enterobacter aerogenes ATCC 13048
Enterobacter amnigenus ATCC 51816
Enterococcus faecalis ATCC 14506
Escherichia coli ATCC 25922
Escherichia coli O157:H7 PSU 93.0134
Escherichia hermanii TICC 2489
Escherichia vulneris TICC 447
Hafnia alvei ATCC 51815
Klebsiella oxytoca ATCC 51817
Klebsiella pneumoniae ATCC 23357
Morganella morganii ATCC 25830
Pantoea spp TICC 4488
Proteus mirabilis ATCC 43071
Proteus vulgaris ATCC 13315
Providencia rettgeri TICC 4472
Serratia ficaria TICC 4475
Serratia liquefaciens ATCC 51814
Serratia marcesens ATCC 14041
Shigella sonnei ATCC 25931
Staphylococcus aureus ATCC 25923
Yersinia enterocolitica ATCC 23715
Yersinia kristensenii ATCC 33639
Yersinia ruckerii ATCC 29473
Listeria grayi ATCC 19120
Listeria grayi ATCC 25401
Listeria grayi ATCC 25402

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3M™ Molecular Detection Salmonella – Performance Summary (cont.)
Appendix 2 – Non-Salmonella exclusivity test strains (cont.)

Genus Species/ Serotype ID

Listeria grayi ATCC 25403
Listeria grayi ATCC 700545
Listeria innocua 3M 19B
Listeria innocua 3M 21A
Listeria innocua ATCC 33090
Listeria innocua ATCC 33091
Listeria innocua ATCC 51742
Listeria ivanovii ATCC 19119
Listeria ivanovii ATCC 49953
Listeria monocytogenes ATCC 19111
Listeria monocytogenes ATCC 19112
Listeria monocytogenes ATCC 19113
Listeria monocytogenes ATCC 19116
Listeria monocytogenes ATCC 19117
Listeria monocytogenes ATCC 19118

Abbreviations: TICC - Tecra International Culture Collection; ATCC - American Type Culture Collection; NTCT- National Collection of
Type Cultures; PSU – Penn State University; ILSI – International Life Science Institute; others - isolates from naturally contaminated
samples

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3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
Appendix 3 –List of Foods
Meat
Turkey breast, raw, ground
Turkey, raw, ground
Chicken, raw, ground
Chicken, frozen nuggets
Chicken, crispy breast strips
Breaded nuggets (chicken substitute)
Beef, raw, ground, 80% lean
Beef, raw, ground, 85% lean
Beef, raw, ground, Angus steak
Seafood
Breaded fish sticks, minced
Frozen beer-batter fillets
Grilled fillets lemon pepper
Cooked frozen shrimp
Shrimp, canned
Shrimp, raw frozen
Spices and Condiments
Paprika
Black pepper
Parsley flakes, dried
Cinnamon
Oregano Leaves
Nuts
Hazelnut spread
Sunflower seed spread
Dairy
Milk, instant non fat dry
Milk, fluid, 1% milk fat
Milk, fluid, 4% milk fat
Milk, lactose free, 1%
Buttermilk (dry)
Ice cream, vanilla
Ice cream, butter pecan
Ice cream, toasted almond fudge
Frozen mozzarella sticks
Cheese, processed, Brand A
Cheese, Brand B
Cheese, processed, spread
Produce
Breaded jalapenos stuffed with cheddar
cheese
Mushrooms, breaded fried
Lettuce, hearts, pre-washed
Arugula, bagged, pre-washed
Carrot juice, 100%
Apple juice
Orange juice from concentrate
Orange juice, pulp free
Pet foods
Dog food, dry
Dog food, canned
Dog food
Soft bacon wrapped sweet potato
intermediate moisture dog treat
Egg products
Liquid egg substitute
Egg, raw shell
Egg, pre-cooked, shelled
Egg white, liquid, 100%
Egg (dried whole)
Egg (dried egg white)
Confectionary products
Chocolate bar, 72% cacao
Chocolate, dark chocolate almond
Chocolate covered peanuts
Chocolate, milk chocolate
Peanut butter
Other processed foods
Whey protein
Vanilla nutritional drink
Chocolate nutritional drink
Milk-based infant formula, Brand A
Milk-based infant formula, Brand B
Soy-based infant formula
Cereal, crispy rice
Cereal, golden puffs
Cereal, blueberry
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3M™ Molecular Detection Assay Salmonella – Performance Summary (cont.)
Appendix 4 – Calculation and Interpretation

Chromogenic agar and quantitative-PCR Chromogenic agar and quantitative-PCR

Reponses
Positive (R+) Negative (R-)
3M method Positive (A+) +/+ Positive Agreement (PA) -/+ Positive Deviation (PD) as ( )

3M method Negative (A-) +/- Negative Deviation (ND) as( ) -/- Negative Agreement (NA)

Accuracy (AC), Specificity (SP), and Sensitivity (SE) of the 3M method were calculated as follow:
( )
AC = x 100%

SP = x 100%

SE = x 100%

Where N = total number of samples

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