TML\MSH Microbiology Department Policy & Procedure Manual
Contents
1. TML MSH Microbiology Department Policy MI SER 16 v01 Page of 10 Policy amp Procedure Manual Section Serology Manual Subject Title Molecular Testing West Nile Virus RT PCR Issued by LABORATORY MANAGER Original Date March 14 2001 Approved by Laboratory Director Revision Date October 10 2003 WEST NILE VIRUS RT PCR I Introduction I West Nile virus WNV is a flavivirus belonging to the Japanese Encephalitis group The primary host is wild birds and the virus is usually transmitted to humans via mosquitoes Transplants and blood transfusions have also been implicated in its transmission This assay involves isolating the viral RNA using the Qiagen Viral RNA Kit and amplifying it by RealArt WNV Reverse Transcription RT PCR Kit The amplification and detection take place simultaneously continually and in real time in the Roche LightCycler In real time PCR fluorescent dyes are linked to oligonucleotide probes which bind specifically to the amplified product Monitoring the level of fluorescence allows the detection of the amplicon In the Artus WNV assay two fluorescent dyes are incorporated in the master mix to detect two amplified products One for the 80 bb WNV amplicon and the other for the Internal Control IC The IC is added to each specimen in the initial step to both control the isolation procedure and to check for PCR inhibition The LightCycler simultaneously monitors the two differe2. From Qiagen Viral Isolation Kit Collection Tubes Lysis Buffer AVL with RNA carrier thawed or heated to dissolve crystals formed during refrigeration Spin Columns Wash Buffer 1 Buffer AW1 add 125 mL ethanol before use e Wash Buffer 2 Buffer AW2 add 160 mL ethanol before use Elution Buffer Buffer AVE From RealArt WNV LC RT PCR Kit Artus e Quantification Standard no 3 10 copies uL e Internal Control Ethanol anhydrous reagent grade 96 100 GENERAL PRECAUTIONS e Use only filtered pipette tips e Store positive materials specimens controls and amplicons separately from all other reagents and add to reaction mixes in a spatially separated facility clean room e Clean and specimen pipettes and tips stay separated in their designated rooms Thaw components thoroughly at room temperature Mix components and centrifuge briefly Work quickly in the cooling block Put on fresh gloves and clean gown colour coded blue when entering the clean room PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 86 TML MSH Microbiology Department Policy MI SER 16 v01 Page 4 of 10 Policy amp Procedure Manual Serology Manual E Sample Preparation Use Specimen Preparation area work in cabinet with gown and frequent glove change Lysis and Purification Steps a For each specimen Quantification Standard QS
3. PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 87 TML MSH Microbiology Department Policy MI SER 16 v01 Page 5 of 10 Policy amp Procedure Manual Serology Manual b i Repeat Step 7 by pipeting the remaining 630 uL of the mixture to the QIAamp spin column in a 2 mL collection tube close the cap and centrifuge at 6000g 8000 rpm for 1 minute Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate j Open the spin column and add 500 uL of Buffer AW1 Close the cap and centrifuge at 6 000g 8 000 rpm for 1 minute k Open the spin column and add 500 uL of Buffer AW2 Close the cap and centrifuge at full speed 20 000g 14 000 rpm for 3 minutes l Place the spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate Centrifuge at 20 000g for 1 min Elution Steps Place the spin column in the second clean 1 5 mL microcentrifuge tube with the LIS specimen label Discard the collection tube containing the filtrate Open the spin column and add 50 uL of Elution Buffer Buffer AVE Close the caps and incubate at room temperature for 1 minute Centrifuge at 6000g 8000 rpm for 1 min Store the eluate at 20 C or 70 C if not able to proceed immediately PCR Amplification and Detection Procedures In the Clean Room Changed to dedicated
4. DEPARTMENT Page 92 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 16 v01 Serology Manual Page 10 of 10 Daily QCs Every run e Fach patient specimen must have an Internal Control IC added to monitor both isolation and PCR inhibition The sample must show a positive reading in either fluorimeter Channel F1 WNV amplicon or fluorimeter Channel F3 back F1 IC amplicon to be valid no PCR inhibition e A Positive Control eg Quantification Standard 3 is included and shows a positive reading in fluorimeter Channel F1 WNV amplicon e A Negative Control eg H20 spiked with IC is include and shows a negative reading in fluorimeter Channel F1 WNV amplicon and a positive reading in fluorimeter Channel F3 back F1 IC amplicon Report all failed QCs to senior charge technologist VI References QIAamp Viral RNA Mini Kit Handbook 01 99 Qiagen RealArt WNV LC RT PCR Kit User Manual March 2003 Artus LightCycler Roche Diagnostics PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 93
5. External Control and Negative Control H20 prepare and label two 1 5 mL microcentrifuge tubes sterilized RNase free with the corresponding number in the Worklist Label one of them with the lab number LIS label also this microcentrifuge tube will store the eluted RNA the other will be used for processing and will be discarded Also label one QIAamp Spin Column Qiagen with the number on the Worklist Arrange the 2 microcentrifuge tubes and Columns into 3 rows If the Lysis Buffer is stored in the refrigerator and crystals are present heat to dissolve the crystals Add 560 uL Lysis Buffer AVL with RNA carrier into each of the microcentrifuge tubes in one row these will contain the waste Add 5 uL WNV Internal Control to each of the microcentrifuge tubes with Buffer AVL Add 140 uL specimen to the Lysis Buffer Buffer AVL with RNA carrier vortex for 15 seconds Incubate at Room Temperature 15 25 C for 10 minutes Centrifuge for 10 seconds at 2000 g to remove drops from the lids Add 560 uL ethanol 96 100 to the samples vortex for 15 seconds Centrifuge for 10 seconds at 2000 g to remove drops from the lids Pipette 630 uL half the volume of the mixture specimen lysis buffer ethanol to the QIAamp spin column in a 2 mL collection tube close the cap and centrifuge at 6000g 8000 rpm for 1 minute Place the QIAamp spin column into a clean 2 mL collection tube and discard the collection tube containing the filtrate
6. and capillaries back in the Cooling Block and proceed to the LightCycler In the LightCycler room Turn LightCycler and the computer on Click on the LightCycler icon and click run Select selftest skip if already done within 24 hr or Press OK Select Run Experimental File WNYV protocol Type in file name WNV run number year month date Select run Type in the number of samples to run including QS ext QC and H20 Click Edit Sample Later Type in the lab numbers QS ext QC if any and H2O PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 89 TML MSH Microbiology Department Policy MI SER 16 v01 Page 7 of 10 Policy amp Procedure Manual Serology Manual k On the QS row click the arrow down button to change its description from Unknown to STD and enter the number of copies uL eg QS3 100 l Press done m To Print Results go to FLUORESCENCE and click e Fl e Quantification e Print Window m To Print Internal Control go to FLUORESCENCE and click e F3 BACK FI e Print Window PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 90 TML MSH Microbiology Department Policy MI SER 16 v01 Page 8 of 10 Policy amp Procedure Manual Serology Manual IV Reporting Fluorimeter Channel F1 Fluorimeter C
7. clean room gown and gloves work in Cabinet a Take out the necessary number of WNV Master Mix vials 12 tests per vial from the freezer and thoroughly thaw inside the Biosafety Cabinet Place a capillary for each purified sample QS external QC and H20 into the capillary adaptor of the Cooling Block pre cooled to 4 C Pipette 15 uL Master Mix into the reservoir of each capillary Changed out of dedicated clean room gown PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 88 TML MSH Microbiology Department Policy MI SER 16 v01 Page 6 of 10 Policy amp Procedure Manual Serology Manual In the Specimen Processing Room Changed into specimen room gown a Using a separate filter tip each time carefully pipette 5 uL each of purified sample QS3 external QC and H20 already spiked with 0 1 mL internal control per 1 mL H20 directly into the capillary reservoir Immediately close the capillary with a lid Load capillary tubes into LightCycler LC adaptor and centrifuge at 3000 rpm for 15 seconds in LC centrifuge Transfer the LC adaptor to the LC instrument Alternatively if the LC centrifuge is not available centrifuge the capillary in the adaptors for 10 seconds at 2000 rpm 400g to transfer the mixture from the reservoir to the capillary making sure that the engraved numbers on the adaptors correspond to the correct sample Place the adaptors
8. ed for the RNA eluate While still in the Worklist press Enter to go to the Test Screen Press F1 to move curser to Demographic field Press to Order Entry Press No to confirm editing Press F9 Press Print Labels Press No modify record Select label printer for the LIS label Specimens Processing Specimens should be processed as soon as possible after arriving in virology laboratory a EDTA blood The blood should be centrifuged and an aliquot of about 2 mL plasma frozen unless PCR can be performed immediately CSF An aliquot 0 2 2 mL of the CSF should be frozen unless PCR can be performed immediately PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 85 TML MSH Microbiology Department Policy amp Procedure Manual Policy MI SER 16 v01 Page 3 of 10 Serology Manual After processing an aliquot of the left over specimens should be stored frozen at 70 C D Materials Equipments and Facilities Clean Room with dedicated Biosafety Cabinet MIBCT4 freezer MIFTG gowns and gloves Specimen Preparation area with Biosafety Cabinet and microcentrifuge Detection Room for LightCycler Roche LightCycler programmed for Artus WNV LC PCR Microcentrifuge MICT14 1 5 mL microcentrifuge tubes Cooling Block with capillary adaptors pre cooled to 4 C Variable volume pipettes 1 to 20 uL 10 to 200 uL 100 to 1000 uL
9. hannel F3 WNV amplicon back F1 IC amplicon Interpretations Possible PCR inhibition Repeat assay If still ve on both Fluorimeter Channels report Indeterminate Report Negative Preliminary report Positive Repeat assay to confirm If still ve on fluorimeter Channel F1 Report POSITIVE Preliminary report Positive Repeat assay to confirm If still ve on fluorimeter Channel F1 Report POSITIVE Positive results for the WNV amplicon Channel F1 will have the cycle number printed under the Cross Point Column in the chart and the Calculated values will be at least 10 copies uL gt QS4 The curve will be rising exponentially and levels off to a plateau The sample must be repeated for confirmation Internal Control amplicon need not be positive Indeterminate results may be those with a positive signal for the WNV amplicon Channel F1 cycle number printed under the Cross Point Column in the chart but the Calculated values are less than 10 copies uL lt QS4 The curve will be rising at a high cycle number The sample must be repeated PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 91 TML MSH Microbiology Department Policy MI SER 16 v01 Page 9 of 10 Policy amp Procedure Manual Serology Manual Indeterminate results may also be those with no signal for the WNV amplicon Channel F1 and also no s
10. ignal for the IC amplicon Channel F3 back F1 indicating possible PCR inhibition The test must be repeated Negative results for the WNV amplicon Channel F1 will be blank under the Cross Point Column in the chart and the curve will be rather flat The IC amplicon Channel F3 back F1 must be positive to be valid Negative Report Negative by RT PCR This is a research test performed by using RealArt WNV assay Artus Biotech Inc Indeterminate Report Indeterminate by RT PCR This is a research test performed by using RealArt WNV assay Artus Biotech Inc Positive Report POSITIVE by RT PCR This is a research test performed by using RealArt WNV assay Artus Biotech Inc Phone all Report positive results and inform senior charge technologist Record isolate in LIS by F7 as 77wnv Quality Control Equipment QC Every six months Colour Compensation to compensate interference between different fluorochromes should be run to update the Colour Compensation File Reagent QCs Before every lot change of isolation and or master mix kit An External Control external to Artus Biotech is used to monitor the isolation amplification and detection procedures The result must correspond to expected value supplied by the manufacturer Before every lot change of master mix kit All four Quantification Standards QS 1 2 3 and 4 must be run PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY
11. nt amplicons using two fluorimeter channels Collection and Transport Blood samples for WNV PCR should be collected in EDTA purple top vacutainer tube CSF specimens should be collected in a clean sterile container and sent to the laboratory as soon as possible If specimens cannot be transported immediately they should be kept at 4 C If a delay of more than 6 hours is anticipated the CSF specimen should be frozen at 70 C The EDTA samples should be processed within 6 hours of collection plasma separated and refrigerated frozen if delay is more than 6 hours Allow only one freeze thaw cycle NOTE Repeated freezing thawing will reduce test sensitivity and cryoprecipitates may accumulate in the plasma PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 84 TML MSH Microbiology Department Policy MI SER 16 v01 Page 2 of 10 Policy amp Procedure Manual Serology Manual HI Procedure A Worklist Preparation a b p C ro mPoaRaogTp Log on to Softmic Select type i Result ii Worklist iii Pagedown to 86 WNV PCR iv Enter v F12 Pending samples plasma serum or CSF will appear on Worklist Mark received samples using the F5 key Type to print Worklist The numbers on the left column of the Worklist will be used to label the 1 5 mL Eppendoff microcentrifuge tubes and the Qiagen Isolation Columns LIS Label Printing if need
Download Pdf Manuals
Related Search