ERP Micro December 2019

Table 2005.04. Interlaboratory study results for the detection of E. coli O157:H7 in selected foods by the Assurance GDS for E. coli O157:H7 method (original study, 2005) GDS for E. coli O157:H7 and reference methods comparison a Test portions confirmed positive Test portions confirmed negative GDS for E. coli O157:H7 performance, % c Level, MPN/g No. labs No. test portions GDS Reference GDS Reference Chi square b Sensitivity rate d % False negatives e Specificity rate f % False positives g

Orange juice

0.0147

14 14 14 12 12 12 10 10 10

84 84 82 72 71 72 59 60 58

58 84

22 29

26

62 55 82 65 54 72 47 23 58

14.0 37.3 NA h

100 100

0 0

92 — 94

8

0.147

0

—

— i

Control

0

0

82

—

6

Ground beef

<0.003 0.0115 Control 0.0036 0.0919 Control

41 68

7

31

18.9 41.0

98

2 0

97

3 0 3

17

3

100

100

0

0

72

NA

—

—

97

Lettuce

22 51

12 37

37

1.9 3.9 NA

91 98 —

9 2

92 89 95

8

9

11

0

0

58

—

5

a Reference method for orange juice and lettuce was FDA; for ground beef was USDA enrichment and FDA confirmation. b Chi square or test for significant difference between two methods that use different primary enrichments is based on confirmed results and is defined as Χ 2 = N(ABS(AD – BC) – N/2) 2 /(A + B)(C + D)(A + C)(B + D) where N = total number of samples tested by the two methods, A = number of samples positive by test method, B = number of samples positive by reference method, C = number of samples negative by test method, and D = number of samples negative by reference method, Χ 2 > 3.84 indicates a statistically significant difference at the 95% probability level. c Sensitivity and specificity rates for reference method for unpaired samples are by study definition 100%. Incidence of false negatives and false positives are by study definition 0%. d Sensitivity rate is defined as the number of analyzed GDS positive results (i.e., reported by the instrument) that were confirmed to be positive from the GDS enrichment divided by the total number of positives (i.e., number of confirmed positives from the GDS method enrichment for all samples, irrespective of results reported by the instrument), expressed as a percent. e Percent false negatives is 100 – sensitivity rate. Low number of total confirmed positives will result in high false-negative data. f Specificity rate is defined as the number of analyzed GDS negative results (i.e., reported by the instrument) that were confirmed to be negative from the GDS enrichment divided by the total number of negatives (i.e., the number of confirmed negative from the GDS method enrichment for all test portions, irrespective of results reported by the instrument), expressed as a percent. g Percent false positives is 100 – specificity rate. Low number of total confirmed negatives will result in high false-positive data. h NA = Statistical analysis not applicable. Methods give equivalent results. i — = Uninoculated control samples are by definition known negatives in study; sensitivity rates not calculated from controls.

(i) Repeat steps in ( f )–( h ) for all samples using new tips for each row of samples. F.E. Amplification and Detection (b) PCR Procedure Note: Amplification tube prep can also be completed using PPMX; for setup procedures please see the PPMX User Manual (No. 55240BC). Change gloves prior to handling reagents. 1) Prior to initial use, the gel cooling block must be stored in the freezer (-25 to -15°C) for at least 6 h. When frozen the gel cooling block will change color from pink to purple. When not in use the gel cooling block should continue to be stored at -25 to - 15°C until it has turned completely purple, indicating it is ready to use. 2) The 72-well aluminum cooling block is for use with the 576

(f) Carefully remove and discard an adhesive film strip from one row of samples. Remove corresponding film strip from strip of sample wells containing wash solution, ( e ) or ( b ). (g) Load PickPen tips, B ( h ), onto the PickPen tool, B ( c ), ensuring that the tips are firmly in place on the PickPen tool. Extend the PickPen magnets and insert into the first strip of sample wells containing sample with concentration reagent. Stir gently for 30 s while continually moving tips up and down from the surface to the bottom of the well. Gently tap the PickPen tips against the side of the sample wells to remove excess media droplets. (h) Transfer PickPen tips to corresponding sample wells containing wash solution and gently swirl for 5 s (do not release particles into solution). Transfer PickPen tips to the corresponding row of the prepared resuspension plate wells, ( c ). With tips submerged, retract the PickPen magnets and tap gently to release particles into the resuspension buffer. Cover resuspension plate with adhesive strips.

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