DETECTION BY REAL TIME RT PCR OF RIFT

SPECIFIC PROCEDURE









TITRE:









DETECTION BY REAL TIME RT-PCR OF

RIFT VALLEY FEVER VIRUS









WRITTEN BY: Núria Busquets DATE: SIGNATURE

Xavier Abad



APROVED BY: DATE: SIGNATURE



REVIEWED BY: DATE: SIGNATURE









CODE: PE PCR xyz

RANGE OF APPLICATION: BSL3 Biocontainment Unit



Date of distribution:

Num of pages: 4

Num Addenda: 0

Num. controlled copies:









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1. INTRODUCTION.



This procedure explains the detection of Rift Valley Fever virus (RVFV) from samples

by using a highly sensitive and specific molecular technique, the real time RT-PCR of a

segment of RVFV genome, extracted from nasal and oral swabs, but also from whole

blood. This procedure, so, include a viral nucleic acid extraction step from the sample

and a further specific amplification of a segment of viral genome (from fragment L)

corresponding to a 135 bp.





2. DEFINITIONS.



RVFV: Rift Valley fever virus

RRT-PCR: real time RT-PCR





3. RANGE OF APPLICATION.



BSL3: Biosafety Level 3, Biocontainment Unit.





4. PROCEDURE.



1-. Sample preparation. All nasal or oral swabs or whole blood samples has been

previously mixed with Trizol (Invitrogen). Briefly, nasal and oral swabs are cut and

kept frozen in a microtube containing 1 ml Trizol (Invitrogen). Whole blood is mixed

(and lysed) in two ways: 0.2 ml total blood with 0.75 ml of Tris reagent lysis buffer

(SIGMA) or 0.5 ml of total blood were lysed with 1 ml of same lysis reagent.

In the case of tissues, 0.1-0.2 g of each tissue must be mixed immediately after obtention

with 0.5 ml of RNAlater (Ambion) and kept overnight at 2-8C. After removing the

reagent, the treated tissues can be kept at -80C in ultrafreezer upon further extraction. For

performing NA extraction from those tissues, they have to be mixed with 1ml of Tris

reagent lysis buffer (SIGMA). All activities involving inactivation from direct (and so,

potentially infected) samples have to be done with proper personal protection equipment

(see items 5 and 6). After mixing with lysis buffer or RNAlater, samples are considered as

non infectious and can be managed using standard procedures.



2-. Viral RNA extraction from the samples. This extraction is performed by following

the Trizol extraction procedure (PE GN 002-01). RNA is suspended in 60 µL of TE and

3 µL (1/20 of the isolated RNA) will be used for the amplification reaction.

Each extraction to be performed in the laboratory must be identified with an extraction

code in the Viral RNA extraction form (PT PCR 227). This number code will identify

the working protocol related to this run (o serial of tests), containing information about

the tested simples, the protocol followed with the possible occurring deviations and

amendments, the date and information about the person/s in charge of this assay. Once

extraction has been performed, extracted RNA is stored in the fridge if RRT-PCR will

be done immediately or kept at -80ºC ultrafreezer in the BSL3 ultrafreezer room

(CR/1028) if the RRT-PCR technique will be scheduled at next days. The positive





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control corresponds to dilution -9 or -10 of a RVFV RNA stock (around 40 and 4

molecules, respectively) and the negative control is PBS.



3-. Amplification of a section of L segment from RVFV genome by RRT-PCR

3.1. Preparation of the reaction: In order to detect the RVFV genome, a RRT-PCR

has to be performed. This step must be performed in the BSL3 Laboratory of

Bacteriology (CR/1033). In the -20ºC freezer (CR-0348) located in that laboratory are

stored all the reagents needed for the amplification reaction. Firstly, the reaction mix

(using one-Step RT-PCR Master Mix Reagents (Applied Biosystems) has to be

prepared in the BSC CR-0248 following the form Record of RRT-PCR from L

fragment of RVFV (Applied) PT PCR xyz, taking into account the number of samples

to be tested. In this form are written down the primers and prove sequences, the reagent

concentration in the reaction mixture and the amplification conditions in the

thermocycler.

Primers and the TaqMan probe are as follows: RVFV-Fw 5’-ttctttgcttctgataccctctgt- 3’,

RVFV Rv 5’ –gttccacttccttgcatcatctg- 3’ and RVFV probe 5’-FAM-

ttgcacaagtccacacaggcccct-TAMRA-5’.



Segment L (5’-3’) 135bp Final Conc.

Forward primer ttctttgcttctgataccctctgt 600nM

Reverse primerr gttccacttccttgcatcatctg 900nM

Taqman probe FAM-ttgcacaagtccacacaggcccct-TAMRA 250nM





More in detail, 600 nM of RVFV-Fw, 900 nM of RVFV-Rv and 250 nM of RVFV

probe in a final volume of 20 µL were used for each reaction

Each run of amplification must be identified with a univocal code of RRT-PCR in the

form. This numerical identification allow to track the tested samples, the procedure

followed with the possible occurring deviations and incidents, the date and information

about person/s in charge of the test. Once the reaction mix, for the whole number of

samples (including negative and positive controls), has been prepared, 20 µl of this

reaction mix will be dispensed at each well of the AB multiplate for real time

ABI7500FAST device. The extracted viral RNA’s have to be added at each reaction

mixture (well) inside a BSC (usually CR-0249) avoiding cross or back contamination of

the reagents. This BSC will be provided of a vessel (with a cap) containing 300-400 ml

of % Virkon or alternatively 1% household bleach in which all tips in contact with the

samples will be thrown. Once RNA has been added, the multiplate is sealed, inside the

aforementioned BSC, by using a transparent plastic foils designed to work under a real

time ABI7500FAST device. The entire run is accepted as correctly performed in the

case of detection of amplification in the extraction positive control although negative

result in the amplification control has been obtained, as the extraction positive control is

also a control to check proper amplification, and it is not unusual that the amplification

control, as constituted by a RNA molecule, became degraded; the mock sample is water

free from RNAsa and DNasa.



3.2. RRT-PCR run and further analysis of the results: The Real time 7500 FAST

device (CR-0455) is located in the BSL3 Laboratory of Equipments (CR/1034),

following a specific procedure PE EQ 140/01C. The amplification conditions are the

following reverse transcription at 50ºC for 20 min; initial denaturing reaction at 95ºC

for 10 min and 40 PCR-cycles of 95ºC 30 s, 55ºC 1 min and 72ºC 30s.





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5. LEVEL OF RISK (CHEMICAL, BIOLOGICAL...).



Microorganims of Biosafety level 3. All handling or processing with infectious samples

has to be performed wearing and additional Tyvek gown, FFP3 face mask (or

equivalent), double gloves and a Sundström ventilated (positive pressure) overhead

protection SR530 with filter SR510 and ventilator SR500, or equivalent system, in a

closed laboratory restricted to any other activities. Once samples has been inactivated

(using lysis buffer from several extraction kits, Trizol, or thermal treatment), and after

intense laboratory decontamination, which includes carefully decontamination of

external surface of all microtubes, vessels and plastic boxes containing samples, by

using 1% Virkon solution or PERAsafe and 10 minutes contact time, with render

environ and samples non infectious, the further steps can be done using conventional

wearing (lab coat, double gloves) with no restriction to other laboratorial activities.





6. SAFETY MEASURES / WASTE MANAGEMENT.



The initial handling of samples, until buffer lysis or Trizol is added, has to be performed

inside Biological Safety Cabinets (BSC) under special personnel protection equipment.

All single use materials used thorough the NA extraction procedure, although starting

samples are considered as non infectious, has to be disposed inside a biodangerous

container (type III).





7. PERSONNEL QUALIFICATION.



Laboratory technician trained in cell culture or virological techniques or any other

research personnel with high qualification in virology.





8. RESPONSABILITIES.



The responsibility of this procedure is on hands of the Area Responsible person. The

BSL3 Laboratory Manager will be the person in charge of the surveillance and control

of all operations performed in BSL3 Laboratories Area.

The laboratorial technicians are responsible to apply correctly the protocols in use and

to use in a proper way, the facilities, materials, equipment and stick to the biosafety

rules in force.





9. REFERENCES/ FORMS/ ADDENDA.









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