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necessary to use the lysis reagent provided with the test kit for this application. ( 3 ) Transfer the cluster tubes to the 80°C heater block and incubate for 20 min. Note: The incubation time may be extended to a maximum of 60 min for the purpose of managing staggered assay start times. ( 4 ) Approximately 3 min before the end of the lysis step, preheat the ANSR reaction tubes to 56°C by placing the tubes in the incubator/reader. Note: The strip of tubes may be cut to provide the number of tubes needed. ( 5 ) At the end of the 20 min lysis incubation, remove and discard the caps from the reaction tubes. Note : Steps ( 6 )–( 8 ) should be completed without delay (within 1 min). ( 6 ) Using an 8-channel micropipettor and 100 µL tips with filters, carefully transfer 50 µL of the lysed samples to the reaction tubes. Mix by rapidly pipetting up and down at least 10 times until the sample appears homogenous in the pipet tip. Avoid excessive bubble formation by not depressing the pipettor plunger beyond the first stop. ( 7 ) Place the permanent caps on the reaction tubes and close the lid of the incubator/reader. ( 8 ) Click START in the ANSR software to begin the assay. ( 9 ) The assay will complete in 10 min and results will be displayed. F. Interpretation of Results TheANSR software will indicate the test results as POSITIVE, NEGATIVE, or INVALID. A positive result indicates that the colony tested contains Salmonella spp. A negative result indicates that the colony tested does not contain Salmonella spp. Assays producing invalid results must be repeated. The real-time fluorescence curves for both the test and positive control channel can be viewed using the ANSR software. G. Limitations The assay detects serovars of both S. enterica and S. bongori , including all genetic subgroups. In testing of 113 strains of Salmonella spp., representing 108 serovars, only a single strain of S . Weslaco was not detected. A summary of results for inclusive and exclusive isolates is shown in Tables 2013.14A and B , respectively. Detailed results, by collaborating laboratory, are shown in Tables 2 and 3. For inclusive strains, all collaborators reported that all six strains grew on all media and a total of 756 ANSR analyses were performed. There were 755 positive results, for accuracy of 99.9% in identification of presumptive Salmonella spp. colonies. Laboratory 2 reported a negative result for S . Enteritidis on HE agar. There is no obvious explanation for this result. There were a maximum of 756 possible results on exclusive strains. There were 65 cases of reported no growth or lack of distinct isolated colonies. A detailed analysis of results showed that three collaborators (laboratories 3, 12, and 13) reported no growth for the Enterobacter cloacae culture on all seven media, accounting for 21 of the no-growth results. Most collaborators reported that neither Providencia alcalifaciens nor Proteus Results

from various suppliers. Follow manufacturer’s instructions for preparation. ( i )  Tryptic soy agar (TSA).— Available from Neogen Corp. and other suppliers. Follow manufacturer’s instructions for preparation. C. Apparatus ( a )  Incubator/reader.— Available from Neogen Corp. Incubator/reader capable of operating at 56 ± 1°C and reading fluorescence in real time in two channels (485/535 nm and 540/590 nm). ( b )  Computer and ANSR software.— Available from Neogen Corp. For connection to incubator/reader. Minimum requirements for computer: Intel ® Core i3 processor, 1 GB RAM, Windows ® 7, Ethernet, and USB connections. ( c )  Heater block.— With insert for 1.2 mL cluster tubes, 80 ± 2°C. ( d )  Micropipettor.— 50 µL, fixed or adjustable volume. ( e )  Pipettor.— 100–1000 µL, adjustable volume. ( f )  8-Channel micropipettor.— 20–200 µL, adjustable volume. ( g )  Pipet tips.— 100 µL, with filter. ( h )  Pipet tips.— 1000 µL. ( i )  Tubes.— Glass or plastic, 12 × 75 mm or similar, sterile, with caps. ( j )  Inoculating loops or needles.— Sterile. D. Preparation of Test Samples Pick an isolated colony from nonselective or selective/ differential agar medium (one of the media listed in section B ) with an inoculating loop or needle and resuspend (vortex or otherwise thoroughly mix) in 0.5 mL PBS in a sterile, capped tube. E. Test Procedure ( a )  General preparation.— ( 1 ) This assay should be performed in a controlled laboratory environment. ( 2 ) Do not use culture media or ANSR reagents beyond their expiration dates. Do not interchange reagents between ANSR kit lots. ( 3 ) Remove ANSR reaction tubes from the foil pouch just before use. Avoid prolonged exposure to light. Tap reaction tubes on bench top to make sure that lyophilized reagents are at the bottom of the tube prior to adding the lysed sample. ( 4 ) Complete all assay steps in sequence, avoiding delays between steps. ( 5 ) Exercise care in pipetting steps to avoid cross- contamination of samples. ( 6 ) Do not remove caps from reaction tubes at any point after the assay is started; this will prevent accidental contamination of the environment with amplification products. ( 7 )  Prior to starting the assay.— ( i ) Preheat the lysis heater block to 80 ± 2°C. ( ii ) Start the ANSR software using the computer connected to the ANSR reader. Select “ Salmonella ” as the test type. Enter sample identifications and other experiment information. The reader will preheat to 56 ± 1°C. ( b )  Assay procedure.— ( 1 ) Add 50 µL of colony resuspension to a 1.2 mL cluster tube. Use a new pipet tip for each sample. ( 2 ) Add 450 µL lysis buffer to the cluster tube. Note : It is not

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