MERS SARS CoV Real TM ver 01042014
Contents
1. Rotor Gene is a registered trademark of Qiagen MX3005P and MX3005P is a registered trademark of Agilent Technologies ABI is a registered trademark of Life Technologies SmartCycler is a registered trademark of Cepheid A D Sacace Biotechnologies Srl a via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 6 4 mail info sacace com web www sacace com 13485 2012 Sacace MERS SARS CoV Real TM VER 01 04 20142. CTION STORAGE AND TRANSPORT MERS SARS CoV Real TM can analyze RNA extracted from nasopharyngeal swabs swab area and place in Eppendorf tube with 0 5 ml of saline water or PBS sterile Sacace Transport medium is recommended Agitate vigorously Repeat the swab and agitate in the same tube Centrifuge at 5000g min for 5 min Discard the supernatant and leave about 100 ul of solution for RNA extraction aspirate bronchial lavage nasal wash centrifuge at 10000 g min for 10 15 min If the pellet is not visible add 10 ml of liquid and repeat centrifugation Remove and discard the supernatant Resuspend the pellet in 100 ul of Saline water plasma whole blood collected in EDTA should be separated into plasma and cellular components by centrifugation at 800 1600 x g for 20 min within six hours The isolated plasma has to be transferred into a sterile polypropylene tube Plasma may be stored at 2 8 C for an additional 3 days Alternatively plasma may be stored at 18 C for up to one month or 1 year when stored at 70 C feces prepare 10 20 feces suspension for instance adding 4ml of Saline Solution and 1 0 gr approx 1 0 ml of feces in 5 ml tube the same can be done in 2 0 ml tube The RNA purification must be done immediately if it is not possible add 20 Glycerol sterile solution cryoprotective agent that provides intracellular and extracellular protection against freezing and store at 20 C Vortex to get an homogeneous suspensio
3. NALYSIS 1 The sample is considered to be positive for MERS if in the channel Rox Orange the value of Ct is different from zero must be less than 33 If Ct value is more than 33 the assay should be repeated and the sample is considered to be positive in case of result s repeat or in case of result is less than 33 2 The sample is considered to be positive for SARS if in the channel Joe Yellow the value of Ct is different from zero must be less than 33 If Ct value is more than 33 the assay should be repeated and the sample is considered to be positive in case of result s repeat or in case of result is less than 33 3 The sample is considered to be negative for MERS amp SARS if in the channel Fam Green value is not determined the fluorescence curve does not cross the threshold line and in the results table on the channel Fam Green the Ct value is lower than 28 Table Results for controls Stage for NCE o i Pos lt 30 Neg Neg Valid result NCA PCR Neg Neg Neg Valid result Pos cDNA PCR Pos lt 28 Pos lt 28 Pos lt 28 Valid result PERFORMANCE CHARACTERISTICS Sensitivity The kit MERS SARS CoV Real TM allows to detect MERS CoV and SARS in 100 of the tests with a sensitivity of not less than 500 copies ml Specificity The analytical specificity of MERS SARS CoV Real TM PCR kit is ensured by selection of specific primers and probes as well as strict reaction conditions The pr
4. _iSacace BIOTECHNOLOGIES For in Vitro Diagnostic Use 6 MERS SARS CoV Real TM Handbook Real Time PCR kit for detection and differentiation of Middle East Respiratory Sindrome MERS CoV and Severe Acute Respiratory Sindrome Coronaviruses SARS REF V65 50FRT NZ 50 Sacace MERS SARS CoV Real TM VER 01 04 2014 Sacace MERS SARS CoV Real TM VER 01 04 2014 NAME MERS SARS CoV Real TM INTRODUCTION Coronaviruses are a large family of ribonucleic acid RNA viruses capable of infecting humans and a number of animal species In humans coronaviruses may cause a range of illnesses from the common cold to severe acute respiratory syndrome SARS Middle East Respiratory Syndrome MERS is viral respiratory illness first reported in Saudi Arabia in 2012 It is caused by a coronavirus called MERS CoV Early reports compared the virus to severe acute respiratory syndrome SARS and it has been referred to as Saudi Arabia s SARS like virus Most people who have been confirmed to have MERS CoV infection developed severe acute respiratory illness As of 31 October 2013 the World Health Organization confirmed that 149 people have contracted MERS worldwide of which 63 have died Affected countries in the Middle East include Jordan Kingdom of Saudi Arabia KSA the United Arab Emirates UAE Oman Kuwait and Qatar in Europe countries affected include France Germany the United Kingdom UK and Italy and in North Afr
5. everse transcription of the RNA e RT G mix 1 5 x 0 01 ml e RT mix 5 0 125 ml e Reverse transcriptase M MLV 0 03 ml e TE buffer 1 2 ml Contains reagents for 60 tests Part N 2 MERS SARS CoV Real TM Real Time amplification kit e PCR mix 1 0 6 ml e PCR mix 2 FRT 0 3 ml TaqF Polymerase 0 03 ml e Pos cDNA MERS SARSIIC C 0 2 ml e DNA buffer 0 2 ml e Negative Control 1 2 ml e Internal Control RNA IC 5 x 0 12 ml Contains reagents for 55 tests must be used in the isolation procedure as Negative Control of Extraction add 10 ul of Internal Control RNA during the RNA purification procedure directly to the sample lysis mixture Sacace MERS SARS CoV Real TM VER 01 04 2014 MATERIALS REQUIRED BUT PROVIDED Zone 1 sample preparation e RNA extraction kit e Biosafety cabinet e Desktop microcentrifuge for eppendorf type tubes e 60 C 5 dry heat block e Vortex mixer e Pipettes with aerosol barrier e 1 5 ml polypropylene sterile tubes Sarstedt QSP Eppendorf e Disposable gloves powderless e Tube racks e Refrigerator e Freezer Zone 2 RT and amplification e Real Time Thermalcycler e Workstation e Pipettes with aerosol barrier e Tube racks STORAGE INSTRUCTIONS MERS SARS CoV Real TM must be stored at 20 C The kits can be shipped at 2 8 C for 3 4 days but should be stored at 20 C immediately on receipt STABILITY MERS SARS CoV Real TM i
6. ica Tunisia Infections presumably acquired through exposure to non human sources have all occurred in the Middle East limited transmission in the countries of Europe and North Africa has occurred in close contacts of recent travellers from the Middle East INTENDED USE MERS SARS CoV Real TM is Real Time PCR test for the qualitative detection and differentiation of Middle East Respiratory Sindrome MERS CoV and Severe Acute Respiratory Sindrome Coronaviruses SARS PRINCIPLE OF ASSAY MERS SARS CoV Real TM Test is based on three major processes isolation of virus RNA from specimens reverse transcription of the RNA Real Time amplification of the cDNA Coronavirus detection by the polymerase chain reaction PCR is based on the amplification of pathogen genome specific region using specific primers and detection via fluorescent dyes These dyes are linked with probes of oligonucleotides which bind specifically to the amplified product The real time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run MERS SARS CoV Real TM PCR kit is a qualitative test which contain the Internal Control IC It must be used in the isolation procedure in order to control the process of each individual sample extraction and serves also to identify possible reaction inhibition Sacace MERS SARS CoV Real TM VER 01 04 2014 MATERIALS PROVIDED Part N 1 Reverta L R
7. imers and probes were checked for possible homologies to all sequences deposited in gene banks by sequence comparison analysis Specificity was confirmed on the following microorganism strains Coronaviruses Cov E229 Cov OC43 Cov HKUI Cov NL63 FCO FC1 CCV Enterovirus strains Adenovirus influenza virus A and B rhinovirus RS viruses Parainfluenza Virus Streptococcus spp Staphylococcus aureus Mycoplasma pneumoniae Chlamydophila pneumoniae Haemophilus influenzae Moraxella catarrhalis Legionella pneumophila Specificity of the kit was confirmed testing positive control samples of RNA MERS CoV hCov EMC upE Assay IVT RNA recommended by WHO for screening tests Institute of Virology University of Bonn Medical Centre Germany and positive control samples of SARS RNA University of Frankfurt Germany Sacace MERS SARS CoV Real TM VER 01 04 2014 TROUBLESHOOTING 1 Weak or absent signal of the IC Fam Green channel retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended RNA extraction method and follow the manufacturer s instructions e The reagents storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Ma
8. ke attention during the RNA extraction procedure 2 Weak signal on the Joe Yellow Cy3 HEX and Rox TexasRed channels retesting of the sample is required 3 Joe Yellow Cy3 HEX or Rox TexasRed signal with Negative Control of extraction e Contamination during RNA extraction procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips among tubes Repeat the RNA extraction with the new set of reagents 4 Any signal with Negative PCR Control e Contamination during PCR preparation procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive controls at the end Repeat the PCR preparation with the new set of reagents Sacace MERS SARS CoV Real TM VER 01 04 2014 KEY SYMBOLS USED List Number Cautionl Contains sufficient LOT Lot Number for lt n gt tests For in Vitro Diagnostic IVD 9 VER Version Use Negative Control of Store at NCA oe Amplification Negative control of Manufacturer NCE gay Extraction Cj Consult instructions for C Positive Control of use Amplification 0 Expiration Date Internal Control SaCycler is a registered trademark of Sacace Biotechnologies CFX and iQ5 are registered trademarks of Bio Rad Laboratories
9. n and centrifuge for 5 min to 7000 12000g Use the supernatant for the extraction of the viral DNA RNA tissue 1 0 gr parenchimatous organs trachea lung brain homogenized with mechanical homogenizer or scalpel glass sticks teflon pestles and dissolved in 1 0 ml of saline water or PBS sterile Vortex vigorously and incubate 30 min at room temperature Transfer the supernatant into a new 1 5 ml tube Specimens can be stored at 2 8 C for no longer than 12 hours or frozen at 20 C to 80 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace MERS SARS CoV Real TM VER 01 04 2014 RNA ISOLATION Any commercial RNA DNA isolation kit if IVD CE validated for the specimen types indicated herein at the SAMPLE COLLECTION STORAGE AND TRANSPORT paragraph could be used Sacace Biotechnologies recommends to use the following kits gt DNA RNA Prep Sacace REF K 2 9 gt SaMag Viral Nucleic Acids Extraction kit Sacace REF SM003 Please carry out the RNA extraction according to the manufacturer s instructions Add 10 ul of Internal Control during the DNA isolation procedure directly to the sample lysis mixture RT AND AMPLIFICATION Reverse Transcription 1 Prepare Reaction Mix for 12 reactions add 5 0 pl RT G mix 1 into the tube containing RT mix and vortex for at least 5 10 seconds centrifuge briefly Thi
10. n your instrument as follows Rotor type instruments Plate type instruments Step Temperature C Time Cycles Temperature C Time Cycles 1 95 15 min 1 95 15 min 1 95 10s 95 10s 2 54 20s 10 54 25 5 10 72 10s 72 25 5 95 10s 95 10s 20s 25s 3 54 Fluorescence 35 54 Fluorescence 35 detection detection 72 10s 72 25 5 1 For example Rotor Gene 3000 6000 Q Corbett Research Qiagen 2 For example SaCycler 96 Sacace CFX iQ5 BioRad Mx3005P Mx3000P Agilent ABI 7300 7500 StepOne Real Time PCR Applied Biosystems SmartCycler Cepheid MERS Cov is detected on the Rox Orange channel SARS is detected on the JOE Yellow channel C on the FAM Green channel INSTRUMENT SETTINGS Rotor type instruments Calibrate Gain More Seitings Optimisation Threshold Outlier Removal Sepa GENCE FAM Green from 5 Fl to 10 FI 0 1 10 off JOE Yellow from 5 FI to 10 FI 0 1 10 off Rox Orange from 5 FI to 10 FI 0 1 10 off Plate or modular type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline otherwise the threshold level should be raised Set the threshold at a level where fluorescence curves are linear and do not cross curves of the negative samples Sacace MERS SARS CoV Real TM VER 01 04 2014 RESULTS A
11. s mix is stable for 1 month at 20 C Add 6 ul M MLV into the tube with Reagent Mix mix by pipetting vortex for 3 sec centrifuge for 5 7 sec must be used immediately after the preparation If it is necessary to test less than 12 samples add for each sample N in the new sterile tube 10 ul of RT G mix 1 with RT mix and 0 5 N wl of M MLV 2 Add 10 ul of Reaction Mix into each sample tube 3 Pipette 10 RNA samples to the appropriate tube Carefully mix by pipetting 4 Place tubes into thermalcycler and incubate at 37 C for 30 minutes 5 Dilute 1 2 each obtained cDNA sample with TE buffer add 20 pl TE buffer to each tube cDNA specimens could be stored at 20 C for a week or at 70 C during a year Real Time amplification Reaction Mix 25 ul 1 Prepare required quantity of tubes or PCR plate 2 Prepare for each sample in the new sterile tube 10 N pl of PCR mix 1 5 N pl of PCR mix 2 FRT and 0 5 N of TaqF Polymerase 3 Add 15 pl of Reaction Mix into each tube 4 Add 10 ul of cDNA sample to appropriate tube with Reaction Mix Prepare for each panel 4 controls e add 10 ul of DNA buffer to the tube labeled Amplification Negative Control NCA e add 10 ul of extracted DNA of Neg Control to the tube labeled Negative Extraction Control NCE e add 10 pl of cDNA MERS SARS IC C to the tube labeled Cpospna Sacace MERS SARS CoV Real TM VER 01 04 2014 Amplification 1 Create atemperature profile o
12. s stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace MERS SARS CoV Real TM VER 01 04 2014 WARNINGS PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following Clinical specimens from MERS cases should be considered as biological substances category B Use sterile pipette tips with aerosol barriers and use new tip for every procedure Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area Thaw all components thoroughly at room temperature before starting an assay When thawed mix the components and centrifuge briefly Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit af
13. ter its expiration date Dispose of all specimens and unused reagents in accordance with local authorities regulations Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into contact rinse immediately with water and seek medical advice immediately Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed Sacace MERS SARS CoV Real TM VER 01 04 2014 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics Use of this product should be limited to personnel trained in the techniques of DNA amplification Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiration dates printed on the box and labels of all components Do not use a kit after its expiration date SAMPLE COLLE
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