Mycoplasma pneumonia - bio
Contents
1. e Washing Solution 2 50 ml e Sorbent 1 25 ml e DNA eluent 5 0 ml Contains reagents for 50 extractions Part N 2 Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM Real Time Amplification e PCR mix 1 55 ready to use single dose test tubes e PCR mix 2 Flu 0 77 ml e Positive Control Chlamydia pneumoniae C 0 1 ml e Positive Control Mycoplasma pneumoniae C 0 1 ml e Negative Control C 1 2 ml e Internal Control IC human DNA 0 2 ml e DNA buffer 0 5 ml Contains reagents for 55 tests Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation e DNA extraction kit Module No 1 e Biological cabinet e Desktop microcentrifuge for eppendorf type tubes e 60 C 2 C dry heat block e Vortex mixer e Pipettors capacity 5 40 ul 40 200 ul 200 1000 ul with aerosol barrier e 1 5 ml polypropylene sterile tubes Sarstedt QSP Eppendorf e Biohazard waste container e Refrigerator e Freezer Zone 2 Real Time amplification e Real Time Thermal cycler e Reaction tubes e Workstation e Pipettes adjustable e Sterile pipette tips with filters e Desktop centrifuge with rotor for 1 5 2 0 ml tubes e Vortex mixer e Freezer refrigerator STORAGE INSTRUCTIONS Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM must be stored at 2 8 C DNA sorb B must be stored at 2 8 C The kits can be s2. 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 Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number Caution Contains sufficient LOT Lot Number for lt n gt tests Store at VER Version Manufacturer NCA Negativ a Control of Amplification Consult instructions for Negative control of NCE use Extraction Positive Control of Expiration Date ce Amplification nE E E RUO For Research Use Only IC Internal Control SaCycler is a registered trademark of Sacace Biotechnologies iCycler 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 Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013
3. 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 Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM can analyze DNA extracted with DNA Sorb B from e Whole blood collected blood in ACD or EDTA tubes 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 Vortex vigorously and incubate 30 min at room temperature Transfer the supernatant into a new 1 5 ml tube e bronchial lavage centrifuge 10 mL at 3000 g min for 10 15 min Remove and discard the supernatant If the pellet isn t visible add 10 ml of liquid and repeat centrifugation remove and discard the supernatant Resuspend the pellet in 100 ul of saline water e swabs insert the swab into the nuclease free 1 5 ml tube and add 0 2 mL of Transport medium Vigorously agitate swabs in medium for 15 20 sec Specimens can be stored at 2 8 C for no longer than 12 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 DNA ISOLATION The following kit is recommended gt DNA Sorb B Sacace REF K 1 1 B Please ca
4. signal of the IC for the Negative Control of extraction e The PCR was inhibited 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 Check the storage conditions e The PCR conditions didn t comply with the instructions 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 during the pipetting of reagents Make attention during the DNA extraction procedure 2 Weak 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 FAM Green or Rox Orange 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
5. 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 metal tubing for reagent transfer Only for Module No 2 Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 PRODUCT USE LIMITATIONS Use of this product should be limited to personnel trained
6. 10 63 45s 10 72 10s 72 20s 95 10s 95 15s 30 s 45s 3 60 fluorescent 35 60 fluorescent 35 signal detection signal detection 72 10s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example SaCycler 96 Sacace iQ iCycler iQ5 BioRad Fluorescence is detected at the 2nd step of Cycling 2 stage 60 C in Fam Green Rox Orange and Joe Yellow fluorescence channels Mycoplasma pneumoniae is detected on the FAM Green channel Chlamydophila pneumonia on ROX Orange and IC DNA on the JOE Yellow HEX Cy3 channel Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 INSTRUMENT SETTINGS Rotor type instruments Important For the Rotor Gene 6000 must be used software 1 7 Build 67 or updated version for software information contact info sacace com Channel Sanne aa Threshold peices l Slope Correct FAM Green from 5 FI to 10 FI 0 05 5 Off ROX Orange from 5 FI to 10 FI 0 1 5 Off JOE Yellow from 5 FI to 10 FI 0 1 0 Off Plate 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 Results for controls Control SEE o
7. Interpretation control NCE DNA isolation Neg Pos Neg Valid result NCA Amplification Neg Neg Neg Valid result Chl fin pneum C Amplification Neg Neg Pos lt 33 Valid result Myc Pe pneum C Amplification Pos lt 33 Neg Neg Valid result IC DNA Amplification Neg Pos lt 31 Neg Valid result Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of the primers and probes was validated with negative samples They did not generate any signal with the specific Mycoplasma pneumoniae and Chlamydia pneumoniae primers and probes The specificity of the kit Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM was 100 The potential cross reactivity of the kit Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM was tested against the group control It was not observed any cross reactivity with other pathogens Analytical sensitivity The kit Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM allows to detect Mycoplasma pneumoniae and Chlamydia pneumoniae DNA in 100 of the tests with a sensitivity of not less than 500 copies ml The detection was carried out on the control standard and its dilutions by negative sample Target region Mycoplasma putative lipoprotein Chlamydophila ompA Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or no
8. hipped at 2 8 C but should be stored at 2 8 C immediately on receipt STABILITY Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM is 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 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 Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS 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 specimens and reagents Thoroughly
9. iSacace BIOTECHNOLOGIES Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM Handbook Real Time PCR kit for qualitative detection of Mycoplasma pneumonia and Chlamydophila pneumoniae REF B42 4 50FRT REF TB42 4 50FRT 50 Sacace Mycoplasma pneumon iae Chlamydophila pneumoniae Real TM VER 21 03 2013 NAME Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM INTRODUCTION Mycoplasma pneumoniae is spread through respiratory droplet transmission Once attached to the mucosa of a host organism M pneumoniae extracts nutrients grows and reproduces Attachment sites include the upper and lower respiratory tract causing pharyngitis bronchitis and pneumonia The infection caused by this bacterium is called atypical pneumonia because of its protracted course and lack of sputum production and wealth of extra pulmonary symptoms Chlamydophila formerly Chlamydia pneumoniae causes mild pneumonia or bronchitis in adolescents and young adults Older adults may experience more severe disease and repeated infections Approximately 50 of young adults and 75 of elderly persons have serological evidence of previous infection The pathogen is estimated to cause 10 20 of community acquired pneumonia cases among adults The estimated number of cases of C pneumoniae pneumonia is 300 000 cases per year INTENDED USE Kit Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM is a Real Time Amplificatio
10. n test for the qualitative detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in the biological materials whole blood tissue swabs etc PRINCIPLE OF ASSAY Kit Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM is based on two major processes DNA is extracted from samples and amplified using real time amplification with fluorescent reporter dye probes specific for Mycoplasma pneumoniae Chlamydia pneumoniae and Internal Control IC Test contains an IC which serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition IC is detected in a channel other than the M pneumoniae or C pneumonia Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B42 4 50FRT Part N 2 Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM Real Time Amplification e PCR mix 1 55 ready to use single dose test tubes e PCR mix 2 Flu 0 77 ml e Positive Control Chlamydia pneumoniae C 0 1 ml e Positive Control Mycoplasma pneumoniae C 0 1 ml e Negative Control C 1 2 ml e Internal Control IC human DNA 0 2 ml e DNA buffer 0 5 ml Contains reagents for 55 tests Module No 2 Complete Real Time PCR test with DNA purification kit TB42 4 50FRT Part N 1 DNA Sorb B isolation of DNA from clinical specimens e Lysis Solution 15 ml e Washing Solution 1 15 ml
11. rry out DNA extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION 1 ar wo Nn o NoD 10 11 12 13 16 17 Lysis Solution and Washing Solution in case of their storage at 2 8 C should be warmed up to 56 C until disappearance of ice crystals Prepare required quantity of 1 5 ml polypropylene tubes Add to each tube 300 ul of Lysis Solution Add 100 ul of Samples to the appropriate tube Prepare Controls as follows e add 100 pl of C Negative Control to labeled Cneg Vortex the tubes and centrifuge for 7 10 sec Vortex vigorously Sorbent and add 25 ul to each tube Vortex for 5 7 sec and incubate all tubes for 10 min at room temperature Centrifuge all tubes for 1 min at 10000g 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 tubes Add 300 ul of Washing Solution 1 to each tube Vortex vigorously and centrifuge for 1 min at 10000g 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 tubes Add 500 ul of Washing Solution 2 to each tube Vortex vigorousl
12. y and centrifuge for 1 min at 10000g 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 tubes Repeat step 11 Incubate all tubes with open cap for 5 min at 65 C Resuspend the pellet in 50 wl of DNA eluent Incubate for 10 min at 65 C and vortex periodically Centrifuge the tubes for 2 min at maximum speed 12000 16000 g The supernatant contains DNA ready for amplification The amplification can be performed on the same day of extraction Sacace Mycoplasma pneumoniae Chlamydophila pneumoniae Real TM VER 21 03 2013 PROTOCOL Reaction volume 25 ul 1 Prepare required quantity of PCR mix 1 tubes for samples and controls 2 Add 7 ul of PCR mix 2 Flu into each tube 3 Add 10 ul of extracted DNA sample to appropriate tube 4 Prepare for each panel 4 controls e add 10 ul of DNA buffer to the tube labeled Amplification Negative Control e add 10 ul of Chlamydia pneumoniae C to the tube labeled ChI Pneum C e add 10 ul of Mycoplasma pneumoniae C to the tube labeled Myc Pneum C e add 10 ul of Internal Control to the tube labeled C Amplification 1 Create a temperature profile on your instrument as follows Rotor type Instruments Plate or modular type Instruments Step Tar slate Time Repeats EE E Time Repeats 1 95 5 min 1 95 5 min 1 95 10s 95 15s 2 63 30s
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