Animal Health Research Institute

Alex food inspection lab

Genetic detection of Noro virus G I , G II . using Pcr technique

Code No. Test Procedure ( )

Version No. 1
Issue Date 25-01-2022
No. of Pages 10

Preparation Revision Approval

Name Dr amr seif Dr / el sendiouny Dr.Ahmed Aiad
Position Head of bacteriological Quality manager Technical manager for
unit speciality
Sign
Date

) ‫( جدول التعديالت‬

‫اعتمادات الوثيقة‬ ‫التاريخ‬ ‫رقم الصفحة‬ ‫بيان التعديل‬ ‫إصدار مراجعة‬

\ ‫إصدار‬
‫مراجعة و اعتماد‬ ‫إعداد‬ ‫مراجعة‬

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Animal Health Research Institute
Alex food inspection lab

CONTENTS

1. Purpose:
2. Scope:
3. Applicability.
4. Environment.
5. Equipment And Media.
6. Precautions.
7. Procedure.

8 Expression of results

9. Retention And Disposal.

10. Method Validation.
11. Test Report
12. Assuring the test result
13. Appendices.
14 Distribution List.

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Animal Health Research Institute
Alex food inspection lab

1. Purpose:
Detection of Noro virus G I , G II according to Noro virus Real Time RT-PCR Kit User Manual
and Iso 15216 -2 : 2019

2. Scope:
This method for determination of Noro virus G I , G II By using pcr technique

3. Applicability.
This procedure shall be applied by all personnel who authorized to carry
out this test.
4. Environment.
 No air currents
 No dusty atmosphere
 Atmospheric confederation .22±2 ℃. and 45-50% humidity
5. Equipments and kits .
5.1. Equipments and media
Molecular biology grade water
Polyethylene glycol (PEG), mean relative molecular mass 8 000.
Sodium chloride (NaCl).
Potassium chloride (KCl).
Disodium hydrogenphosphate (Na2HPO4).
Potassium dihydrogenphosphate (KH2PO4)
Tris base.
Glycine.
Beef extract powder.
Proteinase K.
Pectinase from Aspergillus niger or A. aculeatus.
Chloroform.
n-Butanol.
Sodium hydroxide (NaOH) (≥ 10 mol/l).
Hydrochloric acid (HCl) (≥ 5 mol/l).
Ethylenediaminetetraacetic acid (EDTA) disodium dihydrate.
Real time pcr (step one )
Centrifuge
refrigerator
sterilized tips

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Animal Health Research Institute
Alex food inspection lab

Vortex
Tubes
Lamina flow
Pipette aid

5.2. kits and reagents

1- VIRAL RNA EXTRACTION KIT
2-NV G I Super Mix
NV G II Super Mix

3-RT-PCR Enzyme Mix

4-Molecular Grade Water
5- Internal Control (IC)
HAV Positive Control -6

Analysis sensitivity: 1×103 copies/ml

Note: Analysis sensitivity depends on the sample volume, elution volume, nucleic acid extraction
methods and other factors .If you use the RNA extraction kits recommended, the analysis sensitivity is
the same as it declares. However, when the sample volume is dozens or even hundreds of times greater
than elution volume by some concentrating method, it can be much higher.

6. Precautions.
 Avoid unnecessary talking
 Wear a laboratory coat to protect your cloths
 Do not put any thing in or near your mouth
 Do not lay the pippete down, or allow it to touch any thing, until it has been
sterilized;
 Wash your hands with soap and water.
 Test portion temperature should not exceed 22± 2 ℃.
 Operation should not be carried out in direct sunlight Mouth pipetting is
prohibited.

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 Animal Health Research Institute
Alex food inspection lab

7. Procedure.
7.1 RNA extraction
Sample preparation :
Food surfaces SWAB :
Swab area (maximum 100 cm ²) by cotton swab emersed on PBS , add 10 µl of process control

then immerse swab in tube contain 490 µl lysis buffer , press against wall of tube > repeat this procedure 3-4 times

- rough surface use more than one swab

Fruit and vegetables
- 25 gm cutted to pieces transferred to 400 ml mesh filter bag , add 40 ml TGBE buffer , 30 unit of Pectinase 10 µl of

process control virus material

- Incubate at room temp for 20 with shaking . in acid fruit check ph each 10 min if u need to adjust ph to 9,5 ±0.5
with Naoh for each adjustement increase time by 10 min for maximum 3 time
- Transfer filterate to centrifugation tube use 2 tubes if needed and Centrifugate 10,000 for 30 min at 5 ºc
- Decant supernatant into tube and adjust ph to 7
- Add ,25 volume of 5x PEG /NACL , SHAKING FOR 1 MIN , then incubate at 5°c for 1 hr with constant rocking

- Centrifuge 10,000 for 30 min , discard supernatant , centrifuge 10,000 for 5 min at 5°c , discard supernatant and

resuspend pellet in 500 pbs

- For soft fruit Transfer susp into narrow gauge choloroform resist tube ,add 500 µl choloform /butanol mixture then

incubate at room temp for 5 min

- Centrifuge at 10.000 for 15 min and transfer aqous phase for RNA EXTRACTION
Bottled water :
-add 10 µl of process control virus material , mix sample , filterate under vaccum

- transfer filter into sterile tube add 4 ml of TGBE buffer

-add 10 ml of TGBE Buffer to embty bottle
-shake both tube for 20 min at 60 oscillation
-pool the elute into one tube
- rinse interior wall with 2 ml of TGBE buffer , adjust ph = 7
-centrifugate 4000 for 15 min then transfer concentrate to clean tube and adjust vol to 500 µl with PBS

Retain for RNA Extraction .

BIVALVE SHELL FISH :

-Open shells with sterile knive, clean mud
-dissect digestive gland transfer to clean petridish , chopit until become like paste
-ad 10 µl of process control virus material

-add 2µl of proteinase k , incubate 37 for 60 min with shaking

-secondary incubate by water bath at 60 for 15 min

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Animal Health Research Institute
Alex food inspection lab

-centrifuge 3000for 5 min , decant super natant and retained for RNA
RNA EXTRACTION :

1-Add 1μl of the Internal extraction control RNA (BLUE) to each sample in RNA
lysis/extraction buffer per sample.
2. Complete RNA extraction according to the manufacturer’s protocols.

2 Internal Control
It is necessary to add internal control (IC) in the reaction mix. Internal Control (IC) allows the user to
determine and control the possibility of PCR inhibition.
Add the internal control (IC) 1μl/rxn and the result will be shown in the HEX/VIC/JOE.

preparing of pcr 7.2

WILL PREPARE 2 REACTION TUBE FOR EACH SAMPLE ONE FOR NV GI AND NV GII

The Master Mix volume for each reaction should be pipetted as follows:

※PCR system without HEX/VIC/JOE channel may be treated with 1μl Molecular Grade Water instead of 1μl IC.
1) The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples,
which includes the number of controls, standards, and sample prepared. Molecular Grade Water
is used as the negative control. For reasons of unprecise pipetting, always add an extra virtual
sample. Mix completely then spin down briefly in a centrifuge.
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Animal Health Research Institute
Alex food inspection lab

2) Pipet 20μl Master Mix with micropipets in sterile filter tips to each Real time PCR reaction
plate/tubes. Separately add 5μl RNA sample, positive and negative controls to different reaction
plate/tubes. Immediately close the plate/tubes to avoid contamination.
3) Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes.
4) Perform the following protocol in the instrument:

Quality control:
Negative control, positive control and internal control curve must be performed correctly,
otherwise
the sample results is invalid.

7.4 interpretation of the result :

The following sample results are possible:

8. Retention And Disposal.

1 - If the materials contain +ve cultures or contaminated tools (pipettes) and all
materials used for confirmatory tests must be sterilized by autoclaving at 121 ° C
for 20 minutes
2 - After autoclaving:
a) Glass materials washed by detergent then tap water and distillated water and leave to
dry at room temperature and sterilized in hot air oven to at least 180 °C for one hour.

b) Plastic materials, removed in special container.

9. Method Validation. The test procedure is based on Noro virus Real Time RT-PCR Kit User
Manual and Iso 15216 -2 : 2019 and validation by using reference material to prove that the
test method consistently yields what it is expected using different matrices
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Animal Health Research Institute
Alex food inspection lab

10. Test Report.

The final laboratory report has a form 20-1

11. Assuring the quality of test results

The laboratory applied the quality control program to insure the quality
12.1 Internal quality control :
 Spiked samples
 Repeatability
 Duplicate test

12.2 External quality control :

 Proficiency test

13. Appendixes :

Appendix 1 : Quick Guide – Cel

Quick guide of extraction of DNA

14. Distribution List :

 Technical Manger specialty

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