DIRECCIÓN GENERAL DE SANIDAD DE LA PRODUCCIÓN AGRARIA SUBDIRECCIÓN GENERAL DE SANIDAD E HIGIENE ANIMAL Y TRAZABILIDAD

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1 GUIDELINES FOR THE DETECTION OF AFRICAN HORSE SICKNESS VIRUS BY REAL TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION ASSAY (Agüero et al; 2008) Contents 1. PURPOSE SCOPE REFERENCES ABBREVIATIONS TEST PRINCIPLE SAFETY PRECAUTIONS GENERAL REQUIREMENTS FOR TEST PERFORMANCE Samples Traceability Reagents Amplification kit Primers and Probe Method General Aspects: Protocol Controls Precautions Critical points ASSAY VALIDATION Test validation Sample results interpretation EXTERNAL QUALITY CONTROL TROUBLESHOOTING Nucleic acid extraction PCR assay ANNEX: SPIKED MATRIX (BLOOD) CONTROL PREPARATION Page 1 out of 15

2 1. PURPOSE The purpose of this method is to rapidly detect the specific presence of African horse sickness virus RNA in equine clinical material by a real time reverse transcription polymerase chain reaction assay (RRT-PCR). Currently this method is included in the OIE Chapter of the Manual of Diagnostic Test and Vaccines for the Terrestrial Animals 2017 as a recommended nucleic acid detection test to detect African horse sickness virus (AHSV), as well as in Annex IV to Council Directive 2009/156/EC as last amended by Commission Implementing Decision (EU) 2016/1840, as regards methods for African horse sickness diagnosis. The method described here has been fully validated according to OIE requirements and endorsed by the OIE Biological Standard Commission in September SCOPE The method is applied to specifically detect AHSV RNA in equine samples such as EDTAblood, tissue homogenates and in cell culture supernatants. 3. REFERENCES 3.1. Agüero M., Gómez-Tejedor C., Cubillo MA., Rubio C., Romero E. & Jiménez-Clavero A. (2008). Real time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. J. Vet. Diagn. Invest., 20, Council Directive 2009/156/EC on animal health conditions governing the movement and importation from third countries of Equidae as last amended Regulation (EU) 2017/625 of the European Parliament and of the Council of 15 March 2017 on official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products as last amended ISO/IEC 17025:2017 General Requirements for the competence of testing and calibration laboratories. International Organization for Standardization OIE Chapter Biosafety and biosecurity: standard for managing biological risk in the veterinary laboratory and animal facilities. Manual for Diagnostic Tests and Vaccines for Terrestrial Animals. Page 2 out of 15

3 3.6. OIE Chapter Quality Management in Veterinary testing Laboratories. In: Manual for Diagnostic Tests and Vaccines for Terrestrial Animals OIE Chapter African horse sickness (Infection with African horse sickness virus). In: Manual for Diagnostic Tests and Vaccines for Terrestrial Animals WHO Laboratory Biosafety Manual, Third Edition. WHO, Geneva, Switzerland WHO International Health Regulations, Second Edition. WHO, Geneva, Switzerland. 4. ABBREVIATIONS AHS: Ct: cdna: DNA: dntp: PCR: RNA: RT-PCR: RRT-PCR: ENC: EPC: ANC: APC: SMC: African Horse Sickness threshold cycle complementary deoxyribonucleic acid deoxyribonucleic acid deoxynucleotides polymerase chain reaction ribonucleic acid reverse transcriptase polymerase chain reaction real time reverse transcriptase polymerase chain reaction Extraction Negative Control Extraction Positive Control Amplification Negative Control Amplification Positive Control Spiked Matrix Control Page 3 out of 15

4 5. TEST PRINCIPLE The polymerase chain reaction (PCR) is a powerful molecular technique which allows the specific detection of AHSV RNA by the amplification of a short viral RNA or genome fragment (target sequence) delimited by a specific primer set A first step of viral RNA extraction from the original material to be analysed is required previous to the amplification, which will be the template for the PCR. AHSV RNA template is first converted into a complementary DNA (cdna) using a reverse transcriptase. The cdna is then used as template for exponential amplification by PCR, which consists of repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by DNA polymerase, resulting in the exponential accumulation of specific fragment whose termini are defined by the 5 ends of the primers. The process is specific due to the use of primers, strands of short oligonucleotide sequences, complementary to the nucleic acid to be detected, that serve as a starting point for DNA synthesis by the DNA polymerase. Real-time PCR is an advanced amplification method which allows the automated detection of the amplicon within each cycle of the PCR, reducing the risk of carry-over contamination with increased specificity and in most cases, even sensitivity. In real-time PCR, the incorporation in the reaction mix of a probe labeled with a fluorescent dye enables detection and measurement of the amplicon by using an instrument that combines thermal cycling with fluorescent dye scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction Figure1: Relative fluorescence vs. Cycle number. Amplification plots are created when the fluorescence signal from each sample is plotted against cycle number, therefore, amplification plots represent the accumulation of Edition product 1. January over 2018 the duration of the real-time PCR experiment. The samples used to create this plots are a dilution series of the target DNA sequence. Page 4 out of 15

5 The AHSV real time RT-PCR method uses a primer set and a specific TaqMan MGB probe directed to a highly conserved region of the viral genome, VP7 gene (segment 7), which is able to detect a wide range of AHSV isolates from the 9 known serotypes of the virus. The primers are complementary to the nucleotide positions (forward primer), and to (reverse primer) referred to the AHSV-4 strain (GenBank D12533). TaqMan probe employed for amplified product detection is complementary to nucleotide positions and is labelled with 6-carboxyfluorescein [FAM] at 5 end and brings a minor groove binder (MGB) at 3 end. 6. SAFETY PRECAUTIONS The assay shall be carried out following Biosafety, Biocontainment and Bioprotection guidance, and rules established according to international and national regulations (see references). According to OIE Manual, there is no evidence that humans become infected with any field strain of AHSV, either through contact with naturally or experimentally infected animals or by virus manipulation in laboratories. 7. GENERAL REQUIREMENTS FOR TEST PERFORMANCE 7.1. Samples Sample s biological properties shall be guaranteed by applying correct sampling, preservation and transport procedures until arrival at the laboratory. PCR results might be negatively influenced by poor condition of samples (e.g. putrefaction) EDTA blood samples shall be kept at 4ºC (3-8 days) until they are assayed. Tissues from infected animals can be stored at 4ºC (24-72 hours). For prolonged storage periods; samples shall be frozen at -70 º C. Page 5 out of 15

6 7.2. Traceability The traceability system in the laboratory shall preserve the correct identification of samples during laboratory operations Reagents Amplification kit Some examples of amplification kit to be used are given below: AGPATH-ID ONE-STEP RT-PCR KIT (for fast RRT-PCR) Components: RT-PCR Buffer RT-PCR Enzyme Mix Nuclease-free water QUANTITEC PROBE RT-PCR KIT Components: Quantitec Probe RT-PCR Master Mix Quantitec RT-Mix RNase- free water Primers and Probe Primer Forward: Primer Reverse: Probe: AHS-F1 AHS-R1 AHS-PR1 5 -CCA GTA GGC CAG ATC AAC AG CTA ATG AAA GCG GTG ACC GT FAM-GCT AGC AGC CTA CCA CTA-MGB Method General Aspects: The assay amplifies a RNA fragment of 83 bp of the AHSV VP7 genome region. The PCR is carried out in a final volume of 20 µl. RNA sample is added to each reaction tube containing the primers mix and heat denaturated. Page 6 out of 15

7 One step RT-PCR master mix including Taqman probe is added to reaction tubes after denaturation Protocol Commercial kits to extract viral nucleic acid are widely available. RNA extraction from blood and tissue samples can be performed following manufacturer s instructions. Several one step real-time RT-PCR kits are commercially available, which can be used depending upon local/case-specific requirements and equipment available. Thaw all reagents and store in ice, except the enzyme mix. Maintain an RNAse and DNAse free work environment. Mix all individual reagents thoroughly and spin down. A test plate layout should be designed and loaded into the real time PCR machine software. Using the layout as a guide 2.5 µl of each primer working stock 8 µm (final concentration 1 µm) is added to each well that will contain RNA samples, positive and/or negative controls. The plate is held on ice. Note: PCR plates can be replaced with tubes or strips as appropriate. Reagent µl/sample Working concentration Final reaction mix concentration AHS-F µm 1 µm AHS-R µm 1 µm Sample (RNA) 2 Total Volume 7 Heat denaturation at 95 C for 5 minutes, followed by rapid cooling on ice for further 5 minutes. An appropriate volume of real time one-step RT-PCR master mix for the number of samples to be tested is prepared following manufacturer s instructions. Probe should be included in a final concentration of 0.25 µm (0.1µl of probe working stock 50 µm per sample). 13 µl of master mix is distributed in each well on the PCR plate containing the denatured primers and RNA. Page 7 out of 15

8 Reagent (AgPATH-ID One Step RT- PCR kit) µl/sample Working concentration Final reaction mix concentration RT-PCR Buffer 10 2x RT-PCR Enzyme x AHS-PR µm 0.25 µm RNase free water 2.1 Denatured mix 7 (RNA+primers) Total Volume 20 The plate is placed in a real time thermal cycler programmed with the following profile: Step Cycle step Temperature Time Nº cycles Reverse transcription 48 C 25 min 1x Hot start 95 C 10 min 1x DNA denaturation 95 C 15 sec PCR cycle Primer annealing 55 C* 30 sec 40x Extension 72ºC 30 sec If reagents and thermal cycler allowing fast reactions are employed, then the following program can be used: Step Cycle step Temperature Time Nº cycles Reverse transcription 48 C 10 min 1x Hot start 95 C 10 min 1x DNA denaturation 97 C 2 sec PCR cycle Primer 40x 55 C* 30 sec annealing/extension * Fluorescence data are acquired at the end of the 55 C step. Page 8 out of 15

9 Controls The inclusion of positive and negative controls during the whole process helps to identify the presence/absence of false negative and false positive PCR results respectively. Extraction controls Extraction Controls are used to validate the success of the extraction step (extraction positive control) as well as to test the correct manipulation as to avoid cross contamination (extraction negative control), reducing the chance of obtaining a false negative or a false positive result in the sample. Extraction Positive Control (EPC) should have AHS virus at a concentration producing a Ct around 32, coming from PCR negative clinical specimens, water or buffer spiked with the proper dilution of an AHSV suspension. In case of availability, well characterized positive clinical specimens can be used instead of spiked controls. Water or buffer is normally used as Extraction Negative Control (ENC). Amplification controls Every PCR assay should be validated by incorporating at least a positive control and one negative control into the PCR batch prior to amplification. The use of Amplification Positive Control (APC) helps to exclude the possibility of false negative PCR amplification results, which may be brought about by either the poor quality of one or more of the reaction reagents (e.g. reaction reagent that are out of date or damaged), or by erroneous omission of one of the reagents of the PCR mix. Positive amplification controls comprise a PCR mix prepared with the same volume and concentration of reagents as the PCR mix being used to test specimens, but with the addition of AHSV nucleic acid at a concentration producing a Ct around 32, coming from a positive specimen or from a viral suspension. Amplification Negative Control (ANC) helps to verify that any specific PCR product amplified within the PCRs assay is not the result of contamination, which may be introduced into the PCR batch via contamination of the laboratory environment or contamination of stock PCR mix reagents. Negative amplification controls comprise a PCR mix prepared with the same volume and concentration of reagents as the PCR mix being used to test specimens, but with Page 9 out of 15

10 the addition of a volume of sterile nuclease free water or sterile nuclease free buffer instead of extracted template nucleic acid. After completion of thermocycling, all negative controls should contain no amplification products and the finding of PCR amplification products within any negative control means that the whole assay should be repeated. Spiked matrix control (SMC) In each assay, at least one sample of each matrix type under analysis should be spiked with AHSV suspension at a concentration producing a Ct around 32 to check for any inhibitory matrix effect that could being interfering PCR reaction. The selected sample will be then tested twice. One time as a normal sample and other as a spiked matrix control. To prepare the Spiked Matrix Control (SMC), an aliquot of the sample will be spiked with a volume of the appropriate dilution of an AHSV suspension or a positive specimen. Alternatively, an endogenous or spiked internal control testing the amplification of a different target could be employed in each sample. Please see annex for an example of SMC preparation Precautions Read and follow carefully the complete procedure. Always wear disposable nitrile or latex gloves. RNA should be thawed and stored in ice throughout the reverse transcription preparation and frozen immediately after using. Repeated freeze/thaw of RNA could cause significant degradation in RNA integrity. Keep reagents to the appropriate temperature before and after use. Probes are highly sensitive to light, so they should be manipulated for the minimum time and must be kept always protected from light. Do not pool reagents from different batches. Avoid any contamination of reagents. It is highly recommended to manipulate reagents in a dedicated clean area. Do not use reagents after expiry date Critical points As with traditional PCR, real time PCR reactions can be affected by nucleic acid contamination, leading to false positive results. Page 10 out of 15

11 Some of the possible sources of contamination are: Cross-contamination between samples. Contamination from laboratory equipment. Carryover contamination of amplification products from previous PCRs being this point the most common source of false positive PCR results. Therefore, it is compulsory that personnel working on PCR follow and carry out some strict rules in order to minimize the contamination risk associated to PCR technique: The procedures require experience of working as much as possible under sterile conditions. All steps of sample analysis PCR should be performed in separate locations, using dedicated equipment and material for pre-pcr (sample preparation and extraction), PCR and post-pcr. Personnel must always wear clean disposable gloves while working in the PCR laboratory, that will be replaced frequently and when personnel goes into a different PCR area. The work surface should be decontaminated with the appropriate product before starting. Eventually this step can be followed by a cleaning of the surface with ethanol (allows more rapid drying). PCR reagents shall be stored in a separate freezer and handled in a separated area using material and equipment dedicated that are not in contact with nucleic acid. Use a new sterile RNase filter pipette tip each time that pipetting from a tube containing any sample or RNA/cDNA. If necessary, tubes containing amplified products should be opened and manipulated only in the post-pcr area, where will be discarded. 8. ASSAY VALIDATION 8.1. Test validation The PCR assay will be valid if after completion of thermocycling program: all positive controls (EPC, APC, SMC) should produce amplification curves. Positive control Ct values should be in the range of the previously assigned Ct value ±2. If not, the assay should be repeated. all negative controls (ENC, ANC) should not produce any amplification curve and the finding of PCR amplification curves in any negative control means that the assay Page 11 out of 15

12 should be repeated. If only ANC showed amplification, the new PCR test could be performed using the same extracted nucleic acid, otherwise the sample should be retested from the nucleic acid extraction Sample results interpretation The sample result is NOT VALID if atypical amplification curves are obtained. If this is the case, the assay must be repeated. The sample result is POSITIVE when a typical amplification curve is obtained and the Ct value (cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold) is lower or equal to the defined Ct threshold (35) within 40 PCR cycles (Ct 35). The sample result is INCONCLUSIVE when a typical amplification curve is obtained and the Ct value is higher to the defined Ct threshold (35) within 40 PCR cycles (Ct 35). The sample result is NEGATIVE when a horizontal amplification curve is obtained and does not cross the threshold line within 40 PCR cycles. 9. EXTERNAL QUALITY CONTROL The laboratory shall participate regularly in external proficiency testing schemes. Participation in such scheme is a requirement for accredited laboratories. According to EU rules, European Union National Reference Laboratories have to participate in the Annual Proficiency Test organized by the European Union Reference Laboratory (EU-RL). Proficiency testing material has been well characterized and any spare material, once the proficiency testing has been completed, can be useful to demonstrate staff competence, or to regularly check test performance. Page 12 out of 15

13 10. TROUBLESHOOTING Nucleic acid extraction Critical Point Prevention actions Verification actions Sample Extraction area separated from Use of ENC contamination areas where extracted nucleic acid or (false positive) amplified products are being manipulated Good laboratory practices when managing samples or positives Low extraction efficiency (false negative) controls. Preventive equipment maintenance Periodical equipment verification Proper extraction reagents storage Use of EPC with Ct 32 tolerance threshold ±2Ct Matrix inhibition effect (false negatives) Proper sample preservation Method validation in each matrix Use of SMC or endogenous internal control PCR assay Critical Point Prevention actions Verification actions Sample contamination (false positive) PCR performing area separated from areas where amplified products are being manipulated Use of ANC Good laboratory practices when Low PCR efficiency (false negative) managing extracted nucleic acids Proper PCR reagents preservation: Do not use out of expired date, limited freezing/thawing cycles Primers (proper storage depending on it concentration, limited freezing/thawing cycles) Probe (proper storage protected from light depending on it concentration, limited freezing/thawing cycles) Use of APC with Ct 32 (tolerance threshold ±2Ct; storage at -20ºC avoiding more than 8 freezing/thawing cycles) Page 13 out of 15

14 11. ANNEX: SPIKED MATRIX (BLOOD) CONTROL PREPARATION 1. Collect AHSV uninfected equine whole blood into EDTA tubes. 2. Prepare tubes containing 270 µl each of the uninfected equine blood. 270µl 270µl 270µl 270µl 270µl 270µl 270µl 3. Make serial 10 fold dilutions of the AHSV viral suspension in MEM medium from 10-1 to Add 30µl of each of the AHSV viral suspension dilution into each tube of blood to obtain a higher tenfold dilution in the blood sample. Page 14 out of 15

15 For example: AHSV viral culture suspension 50µl 50µl 50µl 50µl 50µl 50µl 30µl 450µl 450µl 450µl 450µl 450µl 450µl Dilution µl 30µl 30µl 30µl 30µl 30µl 30µl 270µl 270µl 270µl 270µl 270µl 270µl 270µl Dilution After you have spiked the sample with the appropriate volume of the viral suspension mix the tubes by inversion. 6. After mixing, carry out extraction of the viral nucleic acid from the spiked whole blood sample according to your extraction method. 7. Test the nucleic acid according to your RRT-PCR method. Page 15 out of 15