Legionella pneumophila Real TM Quant Eng ver - bio
Contents
1. 13 Centrifuge the tubes for 1 min at maximum speed 12000 16000 g The supernatant contains DNA ready for amplification If amplification is not performed in the same day of extraction the processed samples can be stored at 2 8 C for at maximum period of 5 days or frozen at 209 80 C Sacace Legionella pneumophila Real TM VER 21 03 2013 PROTOCOL 1 Prepare required quantity of PCR mix 1 reaction tubes for samples and controls Add 7 pl of PCR mix 2 on the wax surface 2 3 Add 10 ul of extracted DNA to appropriate tube 4 For each qualitative test prepare the following controls e add 10 pl of DNA buffer to the tube labeled Amplification Negative Control e add 10 ul of QS3 to the tube labeled Amplification Positive Control 5 For each quantitative test prepare the following controls e add 10 pl of DNA buffer to the tube labeled Amplification Negative Control e add 10 pl of QS1 to the tube labeled QS1 e add 10 pl of QS2 to the tube labeled QS2 e add 10 ul of QS3 to the tube labeled QS3 6 Insert the tubes in the thermalcycler Amplification 1 Create a temperature profile on your instrument as follows Rotor type Instruments Plate type Instruments Step de Time Repeats E EE Time Repeats 1 95 15 min 1 95 15 min 1 95 5s 95 5s 2 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 5s 20s 30s 3 56 fluorescent 40 56 fluorescent 40 signal detection signal dete2. Calculate the concentration of Legionella pneumophila DNA in control and samples according to the following C1 pn pwa COp L Ki pn pna Kic X Cic x 2 Cronona cop L quantity of Legionella pneumophila DNA copies in 1 L of water e Kz pn ona cop ml calculated quantity of Legionella pneumophila DNA copies in 1 ml of sample e Kic cop ml calculated quantity of IC DNA copies in 1 ml of sample e Cic cop ml quantity of IC DNA copies in 1 ml of IC according to Data Card e 2 adjustment for sample filtration The calibration curve correlation coefficient R must be more than 0 97 ag 2 The Efficiency value must be in range 0 85 1 15 ANALYTICAL CHARACTERISTICS ANALYTICAL SPECIFICITY Analytical specificity of the primers and probes was validated with complex negative samples They did not generate any signal with the specific Legionella primers and probes ANALYTICAL SENSITIVITY The analytical sensitivity of the Legionella pneumophila Real TM kit was valuated using the Standard DNA of the Legionella pneumophila This Standard was serially diluted in the DNA buffer and tested in 3 replicates 20 times The analytical sensitivity of the kit Legionella pneumophila Real TM was not less than 1000 copies ml Target region mip gene Sacace Legionella pneumophila Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak Ct gt 35 or no signal of the IC Fam Green channel for the Neg Control of extraction and samples
3. e The PCR was inhibited gt Make sure that you use a recommended DNA extraction method and follow to the manufacturer s instructions Re centrifuge all the tubes before pipetting of the extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant Don t disturb the pellet sorbent inhibit reaction e The reagents storage conditions didn t comply with the instructions gt Check the storage conditions e The PCR conditions didn t comply with the instructions gt Check the PCR conditions and select for the IC detection the fluorescence channel reported in the protocol e The IC was not added to the sample only for environmental samples and controls during the pipetting of reagents Make attention during the DNA extraction procedure 2 Weak Ct gt 35 or no signal of the Positive Control e The PCR conditions didn t comply with the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 Joe Yellow signal with Negative Control of extraction e Contamination during DNA 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 between tubes Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative Control of PCR DNA buffer e Contamination during PCR preparation procedure
4. All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive control at last Repeat the PCR preparation with the new set of reagents Sacace Legionella pneumophila Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number Caution Contains sufficient Lot Number for lt n gt tests For in Vitro Diagnostic Bee pS IVD VER Version Use j Store at Expiration Date Consult instructions for Manufacturer use Negative control of Negative Control of C NCA SET Extraction Amplification Positive Control of C Amplification IC Internal Control SaCycler is a registered trademark of Sacace Biotechnologies CFXTM and iQ5 are registered trademarks of Bio Rad Laboratories Rotor Gene is a registered trademark of Qiagen Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Legionella pneumophila Real TM VER 21 03 2013
5. NA eluent 5 ml Contains reagents for 50 extractions Controls e Legionella C 0 5 ml e Negative Control C 1 2 ml e Legionella IC 0 5 ml e DNA buffer 0 5 ml Legionella pneumophila Real TM e PCR mix 1 70 tubes e PCR mix 2 0 77 ml e Standards o QS1 0 06 ml o QS2 0 06 ml o QS3 0 06 ml Contains reagents for 55 tests Sacace Legionella pneumophila Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation DNA extraction kit module n 1 Biological cabinet Vortex 65 C 2 C dry heat block Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g Tube racks Microcentrifuge tubes 1 5 2 0 ml Pipettes with sterile RNase free filters tips Biohazard waste container Disposable gloves powderless Refrigerator Freezer Zone 2 Real Time amplification Disposable gloves powderless Biohazard waste container Refrigerator Freezer Real Time Thermal cycler Workstation Pipettes adjustable Sterile pipette tips with filters Sacace Legionella pneumophila Real TM VER 21 03 2013 STORAGE INSTRUCTIONS Store kit Legionella pneumophila Real TM at 2 8 C The kit can be shipped at 2 25 C but should be stored at 2 8 C immediately on receipt Store DNA Sorb B at 2 8 C STABILITY Legionella pneumophila Real TM is stable up to the expiration date indicated on the kit label The product will maintain performance through the contr
6. _ sacace BIOTECHNOLOGIES For in Vitro Diagnostic Use Legionella pneumophila Real TM Handbook Real Time PCR Kit for quantitative detection of Legionella pneumophila REF TB50 50FRT REF B50 50FRT 2 50 Sacace Legionella pneumophila Real TM VER 21 03 2013 Sacace Legionella pneumophila Real TM VER 21 03 2013 NAME Legionella pneumophila Real TM INTRODUCTION Legionella pneumophila is a thin pleomorphic flagellated Gram negative bacterium of the genus Legionella L pneumophila is the primary human pathogenic bacterium in this group and is the causative agent of legionellosis or Legionnaires disease Legionella pneumophila named in memory of the deceased veterans is ubiquitous to acquatic environments worldwide and resided as an intracellular parasite of amoeba and protozoa provided a link between natural environment and human disease Thus environmental monitoring especially of potable water cooling towers and related sources is a major focus in efforts to control the spread of this disease Since the initial identification of 235 cases in 1976 Legionnaires disease has become recognized as the most common cause of atypical pneumonia in hospitalized patients It is the second most common cause of community acquired bacterial pneumonia with 25 mortality rate INTENDED USE kit Legionella pneumophila Real TM is a test for Real Time qualitative detection of Legionella pneumophila DNA
7. cs Use of this product should be limited to personnel trained in the techniques of DNA amplification EN375 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 COLLECTION STORAGE AND TRANSPORT Legionella pneumophila Real TM can analyze DNA extracted with DNA Sorb B supplied with the Module No 2 from e Sputum bronchial or tracheal lavage must be treated with the following procedure o Collect sputum into 50 mL single use PP tubes with a screw cap o Ina biological safety cabinet homogenize samples after mixing with equal volume of 4 NaOH solution N acetyl L cysteine may be added if required in the amount of 50 70 mg per sample Mix intensely with a tube rotator for 5 20 minutes depending on the density of a sample o Centrifuge samples at 3000 rpm 2800 3000 g for 15 min and carefully discard the supernatant leaving 500 1000 ul in the tube Resuspend sediment and transfer it into a 1 5 ml tube o Centrifuge samples at 12000 rpm for 5 10 min discard the supernatant and use the same 1 5 ml sample tube for DNA isolation from sample sediment e Nasopharyngeal and throat swabs insert the working area of the probe with cotton swab to sterile disposable tube with 500 ul of sterile saline or phosphate buffer solution PBS Broke off the terminal part of the probe or c
8. ction 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example SaCycler 96 Sacace CFX iQ5 BioRad LineGeneK Bioer The results are interpreted through the presence of crossing of fluorescence curve with the threshold line Legionella pneumophila is detected on JOE Yellow channel while the protrombin gene DNA Internal Control is detected on FAM Green channel Sacace Legionella pneumophila Real TM VER 21 03 2013 Qualitative test The analysis results are considered valid only if the control samples results comply the following Results for controls Stage for Ct channel FAM A Control Ct channel JOE Yellow Interpretation control Green C DNA isolation Pos lt 30 Neg OK C DNA isolation Pos lt 30 Pos lt 30 OK DNA Amplification Neg Neg OK buffer QS3 Amplification Pos lt 33 Pos lt 33 OK Quantitative test The analysis results are considered valid only if the control samples results comply the following Results for controls pane Stage for Ct channel FAM Ct channel JOE EEDEN control Green Yellow C DNA isolation Pos Neg OK C DNA isolation Pos Pos OK DNA buffer Amplification Neg Neg OK QS1 Amplification Pos Pos OK QS2 Amplification Pos Pos OK QS3 Amplification Pos Pos OK Sacace Legionella pneumophila Real TM VER 21 03 2013
9. ella pneumophila DNA detection in environmental samples In this case the Internal Control IC is used Legionella pneumophila mip gene DNA amplification is detected on JOE Yellow channel while the IC DNA amplification is detected on FAM Green channel a quantitative test for Legionella pneumophila DNA calculation in water In this case the Internal Control IC is used Legionella pneumophila mip gene DNA amplification is detected on JOE Yellow channel while the IC DNA amplification is detected on FAM Green channel To quantify Legionella pneumophila and Internal Control DNA copies quantitative standards are used A For quantitation of Legionella pneumophila DNA in water samples every sample must be tested two times starting from the extraction procedure The results is given as the average of two results Sacace Legionella pneumophila Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B50 50FRT Controls e Legionella C 0 5 ml e Negative Control C 1 2 ml e Legionella IC 0 5 ml e DNA buffer 0 5 ml Legionella pneumophila Real TM e PCR mix 1 70 tubes e PCR mix 2 0 77 ml e Standards o QS1 0 06 ml o QS2 0 06 ml o QS3 0 06 ml Contains reagents for 55 tests Module No 2 Complete Real Time PCR test with DNA purification kit TB50 50FRT DNA Sorb B e Lysis Solution 15 ml e Washing Solution 1 30 ml e Washing Solution 2 50 ml e Sorbent 1 25 ml e D
10. in the clinical materials sputum aspirate from trachea nasopharyngeal swabs throat swabs bronchoalveolar lavage tissue microorganism cultures and qualitative detection and quantitation of Legionella pneumophila DNA in environmental samples water washes from environmental objects biofilms scrapes ground Sacace Legionella pneumophila Real TM VER 21 03 2013 PRINCIPLE OF ASSAY kit Legionella pneumophila Real TM is a Real Time Amplification test for the qualitative and quantitative detection of Legionella pneumophila in biological materials and in environmental samples Legionella pneumophila Real TM kit can be used as a qualitative test for Legionella pneumophila DNA detection in the clinical materials During the test multiplex real time PCR of Legionella pneumophila mip gene DNA and protrombin gene DNA is performed Protrombin gene DNA is used as endogenous internal control Legionella pneumophila mip gene DNA amplification is detected on JOE Yellow channel while the protrombin gene DNA amplification is detected on FAM Green channel Protrombin gene DNA is a part of human genome DNA and it should be present in adequate amount in DNA sample no less then 10 genomes Both improper storage conditions and poor DNA isolation process can lead to DNA degradation and loss So the endogenous internal control allows not only to control analysis steps but also to estimate sample handling and storage a qualitative test for Legion
11. nly for environmental samples and controls and 300 ul of Lysis Solution 3 Add 100 50 pl of Samples to the appropriate tube 4 Prepare Controls as follows a add 100 pl of C Neg Control provided with the amplification kit into the tube labeled Cneg b add 50 ul of Legionella C and 50 ul of C into the tube labeled Cpos 5 Vortex the tubes and incubate for 5 min at 65 C Centrifuge for 5 7 sec If the sample is not completely dissolved it is recommended to re centrifuge the tube for 5 min at a maximum speed 12000 16000 g and transfer the supernatant into a new tube for DNA extraction 6 Vortex vigorously Sorbent and add 25 ul to each tube 7 Vortex for 5 7 sec and incubate all tubes for 3 min at room temperature Repeat this step 8 Centrifuge all tubes for 30 sec at 5000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes 9 Add 300 pl of Washing Solution 1 to each tube Vortex very vigorously and centrifuge for 30 sec at 5000g Remove and discard supernatant from each tube 10 Add 500 pl of Washing Solution 2 to each tube Vortex vigorously and centrifuge for 30 sec at 10000g Remove and discard supernatant from each tube 11 Repeat step 10 and incubate all tubes with open cap for 5 10 min at 65 C 12 Resuspend the pellet in 50 ul of DNA eluent Incubate for 5 min at 65 C and vortex periodically
12. ns and reagents Thoroughly wash hands afterwards e Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas e Do not use a kit after its expiration date e Dispose of all specimens and unused reagents in accordance with local authorities regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant e 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 e Material Safety Data Sheets MSDS are available on request e Use of this product should be limited to personnel trained in the techniques of DNA amplification e 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 Some components of this kit contain sodium azide as a preservative Do not use A metal tubing for reagent transfer Only for Module No 2 Sacace Legionella pneumophila Real TM VER 21 03 2013 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnosti
13. ol 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 Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results 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 Legionella pneumophila Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following o A Lysis Solution contains guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowed Contact with acid releases toxic gas Xn R 20 21 22 36 37 38 S 36 37 39 e Use sterile pipette tips with aerosol barriers and use new tip for every procedure e Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Use disposable gloves laboratory coats and eye protection when handling specime
14. ut it off to allow dense closing of tube cup Use the suspension for the DNA extraction e Microorganism cultures suspected of Legionella spp resuspend cultures in 0 5 ml of saline solution or phosphate buffer Use 50 ul of suspension for DNA extraction e Tissue 1 0 gr homogenized with mechanical homogenizer or scalpel glass sticks teflon pestles and dissolved in 1 0 ml of saline water or PBS sterile 1 volume of tissue to 1 volumes of saline solution Vortex vigorously and incubate 30 min at room temperature Transfer the supernatant into a new 1 5 ml tube e Water wastewater from water reservoir drinking water 0 5 L of water is preliminary filtered through paper filter with glass funnel After preliminary filtration water is filtered through membrane filter with pore diameter not more than 0 45 um After filtration membrane filter is chopped by sterile scissors to disposable Petri dish and placed by sterile pincers to 1 5 ml tubes with 1 ml of saline solution The tube is incubated at room temperature during 15 20 min periodically mixing on vortex for ensuring of microflora transition in solution Use 50 ul of solution for DNA extraction Sacace Legionella pneumophila Real TM VER 21 03 2013 e Washes from environmental objects are obtained by probe with cotton swab saturated in sterile saline solution Working end of probe with swab is placed in tube with 1 5 ml of saline solution another part of probe is broken off and moved a
15. way Use 50 ul of solution for DNA extraction e Biofilms scrapes from internal surface of water supply industrial and other equipment for example from tray inside air conditioners Scrapes of moist biofilms under water or on the water air interface are obtained by dry sterile probe and scrapes of dried biofilms are obtained by swab saturated in sterile saline solution Working end of probe with swab is placed in 1 5 ml tube with 0 5 ml of saline solution another part of probe is broken off and moved away Use 50 ul of solution for DNA extraction e Ground 100 g transfer the ground 0 4 1 0 g to the tubes of 5 ml with tightly closable lid Add 3 ml of saline solution in each tube mix careful and decant 5 min Supernatant 50 ul is used for DNA extraction Specimens can be stored at 2 8 C for no longer than 48 hours or freeze 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 Legionella pneumophila Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION reagents supplied with the Module No 2 1 Lysis Solution and Washing Solution in case of their storage at 2 8 C should be warmed up to 60 65 C until disappearance of ice crystals Prepare required quantity of 1 5 ml polypropylene tubes including one tube for Negative Control of Extraction and one tube for Positive Control 2 Add to each tube 10 ul of IC o
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