The FEMOFLOR® Real
Contents
1. 5 7 590 TBM 79 Wawa i 5 10 15 20 B6 ctobacillus 7 gt 2 Scale On s 51 3y 6481 w data modes Special biocenosis report Reports 9 Data analysis 9 1 The software analyzes data automatically Software is provided by DT ite DTprime and DT 96 Real time PCR machine manufacturer 9 2 Upon completion of the run software compares actual position of the strip by mean of ROX dye label position relative to position predefined by the operator If there is a mismatch the software suggests rearrangement of the tubes by default In accordance with the operator order can be rearranged If the operator does not accept rearrangement modified data can be saved in a new file 9 3 The quantitative measure logarithm of concentration and diagram are displayed in the line with taxonomic identifier of bacteria detected by FEMOFLOR Real time PCR Kit 9 4 SIC and IC values must be considered when analyzing results 9 4 1 SIC values lower than 4 0 should be considered as due to an insufficient amount of sample see par 3 2 and the sampling procedure must be repeated 9 4 2 IC values lower than 3 5 should be considered as false due to PCR inhibition PCR DNA extraction or sampling procedure must be repeated 9 5 The quantity of DNA logarithm of concentration in positive controls must be higher than 4 0 The qualitative test for Mycoplasma genitalium must be positiv2. requirements To interpret results successfully and robustly a high quality of sample and appropriate conditions of storage transport and handling are required 6 1 2 Material Professional prescription is required to localize the place of sampling urethra cervix or vaginal wall The decision must be based on a patient s complaints and clinical signs and made by the physician in charge Women must not perform hygiene procedures or syringing prior the sampling procedure To interpret results successfully and robustly sample must contain the largest possible number of epithelial cells with minimum amounts of mucus and blood Inappropriate sampling may result in doubtful results and sample collection may need to be repeated 6 1 3 The features of vaginal sampling The sample must be taken prior to physical inspection Speculum can be treated by warm water before the procedure Antiseptics must not be used for speculum treatment The Sample is taken from the lateral or posteroinferior vaginal wall From virginal women sample must be taken from the entrance of vagina or in special cases from the posterior vaginal wall through the hymenal ring 6 1 4 The features of urethral sampling Patient must not urinate within 1 5 2 hours prior to sampling procedure The external urethral orifice must be treated with a swab moistened with sterile physiological saline solution just prior to the sampling procedure In the case of purulent discharge the
3. sample must be taken 15 20 minutes after urinating In the absence of discharge it is necessary to massage urethra with sampling swab or brush Carefully insert the swab into the woman s urethra to a depth of 1 1 5 cm A child s sample must be taken from the external urethral orifice 6 1 5 The features of cervical sampling Remove mucus with a swab prior to sampling and treat the cervix with sterile physiological saline solution Carefully insert sampling swab into the cervix to a depth of 0 5 1 5 cm Avoid contact with vaginal wall when removing the swab 6 1 6 Guidelines for taking sample to transport medium 6 1 6 1 Open the tube 6 1 6 2 Scrape epithelial cells from the corresponding biotope i e vagina urethra cervix with a sterile swab Put the swab into the tube with transport medium and rinse it thoroughly Avoid spraying of solution Remove swab from solution press it to the wall of tube and squeeze the rest of the liquid Throw out the swab Use a new swab if you need to repeat sampling or to take sample from another biotope 6 1 6 3 Close the tube tightly and mark it 6 2 Transport and storage of sample ATTENTION Overall time from the sample intake until analysis must not exceed 24 hours 10 Store samples at 2 8 If sample cannot be delivered to laboratory within 24 hours it can be frozen once 7 Overview of the analysis procedure 7 1 DNA isolation IMPORTANT A DNA isolation kit is not included in FEMOFLO
4. this specific purpose 4 3 Emergency actions Inhalation Inhalation of the Master Mix contained within this kit is unlikely however care should be taken Eye Contact If any component of this kit enters the eyes wash eyes gently under potable running water for 15 minutes or longer making sure that the eyelids are held open If pain or irritation occurs obtain medical attention Skin Contact If any component of this kit contacts the skin and causes discomfort remove any contaminated clothing Wash affected area with plenty of soap and water If pain or irritation occurs obtain medical attention Ingestion If any component of this kit is ingested wash mouth out with water If irritation or discomfort occurs obtain medical attention 5 Instruments and materials required The following instruments and materials are required when using FEMOFLOR Real time PCR Kit Real time PCR thermal cycler the optical thermal and engine calibrations of DT ite DTprime and DT 96 thermal cyclers are non customizable The instruments are calibrated on manufacturing facility in accordance with internal quality control regulations The calibration information and settings for each instrument can be obtained from DNA Technology customer service by request The validation of the adjustable parameters should be performed in accordance with corresponding User manual High speed centrifuge 13000 rpm Microcentrifuge Refrigerator Thermostat temper
5. 4 8 or 16 test in subsequent runs see par 7 4 for reference Set number of samples and specify tubes including negative and positive controls Specify position of strips in a thermoblock see par 7 4 5 for reference Run PCR Refer to table 5 for the cycling program corresponding to FEMOFLOR 4 8 and 16 tests This program is displayed in a start program window 13 Table 5 DT ite DTprime and DT 96 cycling program Optical of Temperature mum Number data block of cycles collection mode 01 889 0 3 ca 3 ESCERIGHI 640 0 15 oe 640 0 15 4 ao 9 5 i e 5 100 Storage Storage Note You can also use the femoflor amplification program 7 3 FEMOFLOR 4 8 16 test upload procedure The following procedure is applicable for DTlight DTprime and DT 96 Real time PCR machines and must be held prior to first PCR run on a given computer Software version 7 3 3 10 or higher 7 3 5 84 Software older than version 7 3 3 10 is not recommended Note The screenshots are taken from 7 3 3 10 7 3 4 0 7 3 4 2 7 3 6 11 versions FEMOFLOR 4 8 or 16 test for use with DTlight DTprime and DT 96 Real time PCR machines is provided by FEMOFLOR Real time PCR Kit manufacturer The upload and synchronization procedure must be held in a Work with device mode as follows 7 3 1 Launch Re
6. DNA genus specific DNA of Lactobacillus spp and genus species specific DNA of each the opportunistic pathogens or flora according to kit format see tables 1 2 3 for the reference To exclude false negatives human DNA SIC quantity is considered 2 Intended use The FEMOFLOR Real time PCR Kit is intended for research and diagnostic applications The DNA Technology FEMOFLOR Real time PCR Kit is intended for the study of vaginal microbiocenosis The FEMOFLORQ Real time PCR Kit is designed to detect the DNA of opportunistic pathogens see tables 1 2 3 for the reference DNA of Lactobacillus sp and human genomic DNA sample intake control SIC supported by 4S1 452 551 552 651 6S2 instruments xx supported by 4M1 4M3 4M6 5M1 5M3 5M6 6M1 6M3 6M6 instruments 3 3 Contents Paraffin sealed PCR mix including PCR mix for total bacterial DNA amplification PCR mix for genus specific amplification of Lactobacillus spp DNA PCR mix for genus species specific amplification of opportunistic flora DNA Taq polymerase solution Mineral oil Positive control FEMOFLOR 4 is used to perform 48 tests including positive and negative controls This kit provides 7 estimates including 5 groups of bacteria SIC and total bacterial DNA The FEMOFLOR Real time PCR Kit is available in three formats depending on the arrangement of microorganisms to be detected FEMOFLOR 8 is used to perform 24 tests including pos
7. General Wahis 53 1030 Brussels Balgium Plone 32 2 722 56 64 ae Positive control 1 tube DESCEND Pet R1 P801 S346EU ec babs European Authorized Represariativa Center 0E A R C Tue Bd General Vigne 53 1090 Brussels Belgium phone 32 2 732 58 54 fax 321 2 j22 80 08 malligabelis ret T2281 Zheleznedorazhnaya ai Bid 20 Prowino Moscow region und Vene 7 8 Carers fax 112 2 73 ___mail obalis 142281 adarazhnaym Bid 20 Protvina region Russia dna bechnalegy r ph ene 960 44 55 21 Real time FEMOFLOR Real time PCR Kit FEMOFLOR 4 Content of the kit Paraffin sealed 24 strips r e Taq polymerase in buffer 4 tubes Mineral oil 4 tubes Positive control 1 tube 2013 01 01 LOT s ual 2013 10 01 Obalis European Authorized Representative Canter D E A R C REP Bd General Wahis 53 1030 Brussels Belgium phone 32 2732 59 54 fax 32 2 732 20 03 ee _ _ _ 142281 Zhelaznocorozhnaya sl Bid 20 Protvina Moscow region Russia www dna technolaqy ru phone 7 495 990 48 58 22 DNA TECHNOLOGY Scietific Production Firm Manufacturer DNA Technology Research amp Production LLC Russia 142281 Moscow Region Protvino 20 Zheleznodorozhnaya Street Phone fax 7 4967 31 06 70 E mail protvino dna technolo
8. P DNA TECHNOLOGY LZ The FEMOFLOR Real Time PCR Kit Available supply options FEMOFLOR 16 12 tests FEMOFLOR 8 24 tests FEMOFLOR 4 48 tests The manual has approved General Director DNA Technology Research amp Production LLC Dmitrovskiy Vladimir The version 260 ibel Date of the statement 30 09 2013 CE Read entire protocol before use 1 1 Introduction The FEMOFLOR Real time PCR Kit is a qualitative in vitro nucleic acid test The FEMOFLOR assay includes following steps DNA extraction Real time PCR and analysis The extraction procedure can be performed with DNA Technology extraction kits see current manual for the reference or appropriate third party extraction kits The implemented PCR method is based on amplification of a target DNA sequence DNA molecules are heat denatured while the cyclic amplification program proceeds Target specific primers bind to the denatured DNA templates in the presence of dNTP s and Taq polymerase Taq polymerase extends the primers thus providing the synthesis of complementary DNA chains and amplification of target DNA sequence To increase the specificity and sensitivity of reaction a paraffin layer separates the PCR mix and Taq polymerase It hampers the admixture of PCR components at low temperatures thus providing a hot start feature which prevents unspecific PCR Real time PCR technology is based on measurement of fluorescence at every cycle of reac
9. R Real time PCR Kit If the sample is taken in a tube with sterile physiological saline solution PREP NA PLUS or PREP GS PLUS kits are recommended for DNA isolation If the sample is taken in a tube from the PREP RAPID kit PREP NA PLUS kit is recommended for DNA isolation Enquire with customer service about compatibility of third party DNA isolation kits with FEMOFLOR Real time PCR Kit Regardless of the kit used for DNA isolation a negative control blank sample that passed all stages of DNA isolation procedure must be prepared 7 1 1 Isolation of DNA from urogenital samples using PREP NA PLUS kit Note If precipitate occurs in the Lysis Solution dissolve it by heating solution up to 65 C for 10 minutes prior to use Use RNase and DNase free pipette tips only 7 1 1 1 Centrifuge the tubes with samples at 13000 rpm for 10 minutes 7 1 1 2 Remove supernatant leaving approximately 100 HL of liquid precipitate liquid fraction IMPORTANT Perform each step of DNA isolation procedure for negative control C in parallel with test samples Add 100 uL of negative control in a new 1 5 ml tube and perform the procedure according to par 7 1 1 3 7 1 1 18 of current manual 7 1 1 3 Add 300 ul of Lysis Solution into each tube Avoid contact of pipette tip with the edges of the tube 7 1 1 4 Close the tubes tightly Vortex the tubes for 3 5 seconds 7 1 1 5 Incubate the tubes at 65 C for 15 minutes Centrifuge the tubes
10. alTime PCR application Choose operator to work with FEMOFLOR Real time PCR Kit Set Work with device mode Select directory for saving the data when adding new operator 7 3 2 Select Copy groups of tests bookmark in the Test menu Select groups of tests bookmark in the Test menu _ Mode Test Preference Help 1 x Protocol Start program New protocol Open Archived data viewer Protocol number 0 Operator name Date B Tube Concentration m xml Import MultiT est Add E ap ml ee Ch Add Test before after Add line 7 3 3 Select line from ini file in the left module of Copy groups of tests window Open Femoflor ini file 7 3 4 Select operator whose directory will be used to copy FEMOFLOR 4 8 or 16 tests in a right module of Copy groups of tests window 7 3 5 Click arrow button The selected test will appear in a right module of window the recommended version can be changed due to software update The current version is available for download at http www dna technology ru support 14 Select operator group of tests ce Oey femoflor 16 TEST femoflor 4 v femoflor 8 The operator for whom the test was copied can work now with FEMOFLOR 4 8 or 16 test 7 4 Routine work with FEMOFLOR 4 8 o
11. at 13000 rpm for 30 seconds to precipitate condensate 7 1 1 6 Add 400 of Precipitation Reagent into the tubes and vortex the tubes for 3 5 seconds 7 1 1 7 Centrifuge the tubes at 13000 rpm for 15 minutes 7 1 1 8 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 1 9 Add 500 ul of Wash Solution N21 to the pellet and shake the tube 3 5 times gently 7 1 1 10 Centrifuge the tubes at 13000 rpm for 15 minutes 7 1 1 11 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 1 12 Add 300 ul of Wash Solution 2 to the pellet and shake the tube 3 5 times gently 7 1 1 13 Centrifuge the tubes at 13000 rpm for 5 minutes 7 1 1 14 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 1 15 Open the tubes and dry the pellet at 65 C for 5 minutes 7 1 1 16 Add 300 ul of Dilution Buffer to the pellet Centrifuge the tubes for 3 5 seconds to collect drops 7 1 1 17 Incubate the tubes at 65 C for 10 minutes Vortex the tubes for 3 5 seconds 7 1 1 18 Centrifuge the tubes for 30 seconds at 13000 rpm to collect drops 11 DNA sample is ready to use the PCR reaction Store the DNA sample at 20 C for one month or at 70 C for one year 7 1 2 Isolation of DNA from urogenital samples using PREP GS PLUS kit Note If precipitates occur in the Lysis Solution or Wash so
12. ature range 50 65 9C PCR tube rack for 1 5 ml tubes PCR tube rack for strips of eight 0 2 ml tubes 1 5 ml PCR tubes Single channel pipettes volume range 0 5 10 ul 5 40 ul 40 200 ul 100 1000 ul RNase and DNase free filtered pipette tips volume range 20 ul 50 ul 200 ul 1000 ul Powder free surgical gloves Disinfectant solution Physiological saline solution 0 9 NaCl Sterile Container for used pipette tips Nucleic acid extraction kit DNA Technology PROBE NA PLUS or PROBE GS PLUS are recommended Software The recommended version of software is 7 3 5 84 software older than version 7 3 3 10 is not recommended Femoflor ini file the recommended version can be changed due to software update recent version of DT 96 software is available for download at http www dna technology ru support 9 6 Samples A sample of epithelial cells is taken from the urethra cervix or posterolateral vaginal wall depending on professional prescription Sampling processing and storage of samples is held in accordance to PROBE NA PLUS or PROBE GS PLUS instruction 6 1 Sampling procedure Sampling is carried out by scraping of epithelial cells from respective biotope The urogenital Sampling is held by sterile swab or brush The sample is then transferred to plastic tubes containing 300 ul of physiological saline solution or in the tubes containing DNA Technology PROBE RAPID solution 6 1 1 General
13. bacterium spr Sne Lept Fuso Mega Veil Dial Lachno Clost Mobi Coryne Peptostrept 7 4 6 Click Apply button in the lower right corner of the Protocol window 7 4 7 The cycling program will be displayed in Start program window 7 4 8 Click Start program button in the lower right corner of the window 7 4 9 Write the name of file and specify directory to save the data The data will be saved 17 to the operator s working directory by default see par 7 3 1 The cycling program 4 RealTime_PCR E 8 Mode Test Preference Help 3 eure Protocol Start program N New protocol Open Save Lal Archived data viewer Program name femoflor 4 Browse New AN Operator L1 N Description SES Date 00 30 00 40 00 20 00 30 600 x5 00 20 00 05 00 05 00 20 690 x 40 Holding 100 Program femoflor 60 Mixture volume 35 a mcl Comments 60 40 20 IR WI Start program 00 10 00 00 20 00 00 30 00 00 40 00 00 50 00 01 00 00 01 10 00 pen block Close block 8 Data collection The optical unit of the Real time PCR machine collects data automatically as the cycling program proceeds The Real time PCR machine detects and interprets results automatically Upon completion of the run a status screen will be displayed and the operator will be prompted to proceed to analysis User manual Chapter 1 Operation par 4 6 o
14. d Liquid waste containing acids or bases must be neutralized before disposal Wear suitable protective clothes and gloves and protect eyes and face Never pipette solutions by mouth Do not eat drink smoke or apply cosmetic products in the work areas Carefully wash hands after handling samples and reagents Dispose of leftover reagents and waste in compliance with the regulations in force Read the User manual provided with the kit before running the assay While running the assay follow the instructions listed in the User manual Do not use the kit after the expiry date provided Only use the reagents provided in the kit and those recommended by manufacturer Do not mix reagents from different batches Do not use reagents from third party manufacturers kits 4 2 Warnings and precautions for molecular biology Molecular biology procedures such as nucleic acids extraction reverse transcription amplification and detection require qualified staff to avoid the risk of erroneous results especially due to the degradation of nucleic acids contained in the samples or sample contamination by amplification products Equip separate areas for the extraction preparation of amplification reactions and for the amplification detection of amplification products Never introduce an amplification product in the area designed for extraction preparation of amplification reactions Wear lab coats gloves and tools which exclusively employed for the ext
15. e and signed as in corresponding line of the table If the quantity of DNA in positive controls is lower than 4 0 and the qualitative test for Mycoplasma genitalium is negative all results of corresponding lot should be considered as false All samples must be reanalyzed 9 6 The quantity of DNA logarithm of concentration in negative controls must not exceed values shown in table 6 If measured amount exceeds these values all results of the corresponding lot should be considered as false and the PCR laboratory must be decontaminated 19 Table 6 Maximum acceptable amount of bacterial DNA in negative controls FEMOFLOR 16 Acceptable quantity of DNA in K Lg Less than 3 5 Less than 2 5 Less than 2 5 Less than 2 5 Less than 2 5 Less than 2 5 Identifier Total bacterial DNA Lactobacillus spp Enterobacterium spp Streptococcus spp Staphylococcus spp Gardnerella vaginalis Prevotella bivia Porphyromonas spp Eubacterium spp Sneathia spp Leptotrihia spp Fusobacterium spp Megasphaera spp Veilonella spp Dialister spp Lachnobacterium spp Clostridium spp Mobiluncus spp Corynebacterium spp Peptostreptococcus spp Atopobium vaginae Mycoplasma hominis Mycoplasma genitalium Ureaplasma urealyticum parvum Candida spp SIC IWIN NJ OY OT CO I NO e Less than 2 5 Less than 2 5 Less than 2 5 Less than 2 5 Less than 2 5 Less than 2 5 Nega
16. f sample each test result measure of quantity and diagram allowing a relative comparison of normal flora and opportunistic pathogens in each sample will be displayed in the right module of the window A qualitative analysis will be performed for pathogenic bacteria The resulting graph will display the dependence of fluorescence intensity on cycle number for each tube Operator can create save and print a report by clicking Report and select Special biocenosis report 18 Instrument panel Report Mode Preference Help d Protocol x Z Data areis 9 RealTime PCR Protocol Operator Date Re amp ul Comments d 7 T Atopobium_vaginae Mycoplasma_hom Mycoplasma_gen Analysis type Biocenose z E 833 H B 1713 Method Curve Shape e Fas 6 2 Lactobacillus 60 Gawa 8 500 c femoflor 16 8000 E Enterobacterium spp 7 500 P nil D4 Streptococcus spp FPE 7000 wt 5 qx B q 2 din 9 Gard Pre Porph 44 EH m is G4 Eubacterium spp 35 O 7 fe 5500 77 Sne LepuFuso 5000 Z 5 Mega Veil Dial f 8 4500 BS Lachno Clost 4000 NCS Mobi Coryne 8 3500 05 Peptostrept EJ f Ureaplasma_spp 3 0 Candida_spp SIG 4 Threshold 522 2 Marker femoflor 16 1 CTM
17. for 3 5 seconds 7 1 2 13 Centrifuge the tubes at 13000 rpm for 1 minute 7 1 2 14 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 2 15 Add 200 ul of Wash Solution 3 to the pellet Vortex the tubes for 3 5 seconds 7 1 2 16 Centrifuge the tubes at 13000 rpm for 1 minute 7 1 2 17 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 2 18 Open the tubes and dry the pellet at 50 for 5 minutes 7 1 2 19 300 yl of Elution Buffer to the pellet Vortex the tubes for 5 10 seconds 7 1 2 20 Incubate the tubes at 50 C for 5 minutes 7 1 2 21 Centrifuge the tubes at 13000 rpm for 1 minute DNA sample is ready to use in the PCR reaction Store DNA samples at 2 8 up to 7 days Repeat steps according to par 7 1 2 20 7 1 2 21 of current protocol prior to use of DNA sample in PCR 7 2 Amplification 7 2 1 Mark strips with paraffin sealed PCR mix for test samples positive control and negative control C NOTE One strip of FEMOFLOR 4 is used for the test of two samples One strip of FEMOFLOR 8 is used for the test of one sample 12 Two strips of FEMOFLOR 16 are used for the test of one sample Example To analyze 2 samples mark 2 strips 1 for the samples and 1 for positive and negative controls when using FEMOFLOR 4 4 strips 2 for the samples 1 for positive control and 1 for negative contro
18. gy ru http www dna technology ru Seller DNA Technology LLC Russia 117587 Moscow 125Zh Varshavskoe highway bld 6 Phone fax 7 495 980 45 55 E mail mail dna technology ru Authorized representative in EU OBELIS S A Registered Address Bd G n ral Wahis 53 1030 Brussels Belgium Tel 32 2 732 59 54 Fax 32 2 732 60 03 E mail mail obelis net http www obelis net The version 260 ibel Date of the statement 30 09 2013 23 C
19. itive and negative controls The kit provides 11 estimates including 9 groups of bacteria SIC and total bacterial DNA FEMOFLOR 16 is used to perform 12 tests including positive and negative controls This kit provides 25 estimates including 23 groups of bacteria SIC and total bacterial DNA The FEMOFLOR Real time Kit formats contents fluorescent chemistry detection channels color tags Table 1 FEMOFLOR 4 Dye label detection channel of vial Color of Color of in a strip Rox buffer paraffin EM total bacterial DNA Gardnerella vaginalis Prevotella bivia Porphyromonas spp Candida spp Lactobacillus spp Colorless Marker Table 2 FEMOFLOR 8 N of vial Dye label detection channel Color of Color of in a strip buffer paraffin total bacterial DNA Lactobacillus spp Enterobacterium spp Streptococcus spp m B Gardnerella vaginalis Prevotella bivia Porphyromonas spp Eubacterium spp Mycoplasma hominis My coplasma genitalium uni Table 3 FEMOFLOR 16 N of vial Dye label detection channel d of oo of ee qM Ioco total bacterial Lactobacillus spp Enterobacterium spp B Streptococcus spp White Staphylococcus spp Gardnerella vaginalis Prevotella bivia Porphyromonas spp Eubacterium spp Sneathia spp Leptotrihia spp Fusobacterium spp Table 3 FEMOFLOR 16 N of vial Dye label detection channe
20. l of on of ee ee ee p ds g ccLe 0 Megasphaera spp 1 Veilonella spp Blue Dialister spp Lachnobacterium spp Clostridium 5 Mobiluncus spp Corynebacterium Spp Peptostreptococcus Spp Blue B Atopobium vaginae Colorless Mycoplasma hominis genitalium Ureaplasma urealyticum parvum B MN 4 Warnings and precautions As part of industrial and personal hygiene and general safety practices avoid all unnecessary exposure to the chemical components of this kit and ensure prompt removal from skin eyes and clothing upon contact Significant health effects are NOT anticipated from routine use of this kit when adhering to the instructions listed in the current manual 4 1 General warnings and precautions Handle and dispose all biological samples as if they were able to transmit infective agents 7 Avoid direct contact with the biological samples Avoid producing spills or aerosol Any material coming in contact with the biological samples must be treated for at least 30 minutes with disinfecting solution or autoclaved for 1 hour at 121 C before disposal Handle and dispose all reagents and all materials used to carry out the assay as if they were able to transmit infective agents Avoid direct contact with the reagents Avoid producing spills or aerosol Waste must be handled and disposed according to adequate safety measures Disposable combustible material must be incinerate
21. l when using FEMOFLOR 8 8 strips 4 for the samples 2 for positive controls and 2 for negative controls when using FEMOFLOR 16 See table 4 for reference Table 4 FEMOFLOR 4 FEMOFLOR 8_ FEMOFLOR 16 Sample 1 Tubes 1 4 Tubes 1 8 Tubes 1 8 strip 1 and strip 2 Sample 2 Tubes 5 8 Tubes 1 8 Tubes 1 8 strip 1 and strip 2 C Tubes 1 4 Tubes 1 8 Tubes 1 8 strip 1 and strip 2 C Tubes 5 8 Tubes 1 8 Tubes 1 8 strip 1 and strip 2 7 2 2 Mix the Taq polymerase solution thoroughly 3 5 sec then spin briefly 1 3 sec 7 2 3 Aliquot 10 ul of Taq polymerase solution into each tube Avoid paraffin layer break 7 2 4 Aliquot 20 ul of mineral oil one drop from 100 1000 ul tip approximately into each tube Close strips tightly 7 2 5 Open the tube of a strip add DNA sample then close the tube before proceeding to the next DNA sample to prevent contamination Use filter tips Aliquot 5 0 ul of DNA sample into each tube of a strip assigned to test samples 7 2 6 Add 5 0 ul of positive control and negative control see par 7 1 for reference into corresponding tubes 7 2 7 Spin strips briefly 1 3 sec 7 2 8 Set strips to Real time PCR instrument Try to place tubes in the center of the thermoblock 7 2 9 Launch RealTime PCR application Upload the corresponding test FEMOFLOR 4 8 or 16 from file before the first run see par 7 3 for reference Add FEMOFLOR
22. lution 1 dissolve it by heating solution up to 50 C for 15 20 minutes prior to use Use RNase and DNase free pipette tips only 7 1 2 1 Centrifuge the tubes with samples at 13000 rpm for 10 minutes 7 1 2 2 Remove supernatant leaving approximately 100 HL of liquid precipitate liquid fraction IMPORTANT Perform each step of DNA isolation procedure for negative control C in parallel with test samples Add 100 uL of negative control in new 1 5 ml tube and perform the procedure according to par 7 1 2 3 7 1 2 21 of current manual 7 1 2 3 Mix Lysis Solution with the Sorbent in a separate tube as follows e 150x N 1 ul of Lysis Solution e 20x N 1 ul of previously resuspended Sorbent N 1 number of samples to be analyzed including C N and one reserve sample 7 1 2 4 Add 170 ul of prepared mixture into each tube 7 1 2 5 Close the tubes tightly vortex the tubes for 3 5 seconds 7 1 2 6 Incubate the tubes at 50 C for 20 minutes 7 1 2 7 Centrifuge the tubes at 13000 rpm for 1 minute 7 1 2 8 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 2 9 Add 200 ul of Wash Solution 1 to the pellet Vortex the tubes for 3 5 seconds 7 1 2 10 Centrifuge the tubes at 13000 rpm for 1 minute 7 1 2 11 Remove supernatant completely Avoid contact of pipette tip with pellet use new tip for each tube 7 1 2 12 Add 200 ul of Wash Solution N92 to the pellet Vortex the tubes
23. r 16 test 7 4 1 Launch RealTime_PCR application Choose operator for whom the test was copied see par 7 3 4 Set Work with device mode 7 4 2 Click Add test button Click Add test RealTime_PCR E xi Mode Test Preference Help Protocol E E apogan L1 3 Open Save EY Archived data viewer Protocol number 0 Operator name y x gt Es Multi est NN Ch Add Test before after gt Add line em Delete line Floating X Clear protocol UnDo C Scientific AutoFill Clear matris field amp Order fill Fee filling x Apply Cancel 7 4 3 Choose FEMOFLOR 4 8 16 test from the drop down menu Choose FEMOFLOR 4 8 or 16 Add test xf Fa emoflor 1 _ femoflor 4 2 standards Values of standard from other protocol AMOUNT US 7 4 4 Specify number of samples Click OK button Specify number of samples Add test Standards Values of standard from other protocol BOUNT 3 Controls Positive L Negative 12 n Deta 7 4 5 Specify tubes Define position of strips in software interface according to position they were set in thermoblock use Clear the field Clear the field and order fil buttons Specify tubes 2 RealTime PCR Mode Test Preference Help CES ees e ZA Cie Add Te
24. raction preparation of the amplification reaction and for the amplification detection of the amplification products Never transfer lab coats gloves and tools from the area designed for amplification detection of the amplification products to the area designed for extraction preparation of amplification reactions The samples must be exclusively employed for certain type of analysis Samples must be handled under a laminar flow hood Tubes containing different samples must never be opened at the same time Pipettes used to handle samples must be exclusively employed for this specific purpose The pipettes must be of the positive dispensation type or be used with aerosol filter tips The tips employed must be sterile free from the DNases and RNases free from DNA and RNA The reagents must be handled under laminar flow hood The reagents required for amplification must be prepared in such a way that they can be used in a single session Pipettes used to handle reagents must be exclusively employed for this specific purpose The pipettes must be of the positive dispensation type or be used with aerosol filter tips The tips employed must be sterile free from the DNases and RNases free from DNA and RNA Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible in order to avoid the possibility of contamination Pipettes used to handle amplification products must be exclusively employed for
25. st before after Lactobacillus lt gt Addline terobacterium_s v reptococcus sp Delete line aphylococcus si x canes Floating Hemel are Scientif cubacterium_spp Sne Lept Fuso Mega Veil Dial Lachno Clost Mobi Coryne Peptostrept opobium vagin Myc hominis Myc genitalium Jreaplasma spp Candida_spp SIC Marker Sample_2 femoflo TBM P Lactobacillus IC terobacterium_s sp _ Bard Pre PorpkF ubacterium spr s AutoFill Clear matrix field Sne Lept Fuso Mega Veil Dial Lachno Clost fm Mobi Coryne ae Click Order for filling Test Preference 00 M oco 17 Open Li Archived data viewer Protocol number 0 Operator name 7 PUES Date 1 2011 Sample 1 femoflor Lactobacillus IC terobacterium s 1 2 d 4 reptococcus sp 5 aphylococcus_s Marker 6 Gard Pre Porpr 7 ubacterium spr 8 Sne Lept Fuso 3 Mega Veil Dial Lachno Clost Mobi Coryne 12 Peptostrept opobium vagin Myc hominis Myc genitalium Jreaplasma spp Candida spp SIC Marker 17 Sample 2 femoflo TBM 2 B3 18 Lactobacillus IC 19 terobacterium_s 20 reptococcus_sr aphylococcus_s Gard Pre Porpr 5 23 u
26. time PCR Kit to the prescribed technical requirements is subject to compliance of storage carriage and handling conditions recommended by manufacturer Contact our customer service by quality issues of FEMOFLOR Real time PCR Kit 115587 Moscow Varshavskoye sh 125g building 6 DNA Technology Phone Fax 7 495 9804555 Technical support 7 800 200 75 15 E mail hotline dna technology ru www dna technology ru 11 Quality policy DNA Technology Research amp Production LLC established the following goals to realize its quality policy focused on customer satisfaction management by improving the quality management system to comply with 1509001 2000 and ISO13485 2003 observation of quality management in manufacturing of IVDD products creation of values for customers maintenance of the best service quality and customer management 12 The project of samples of marks CE A DNA TECHNOLOGY Real time FEMOFLOR DNA TECHNOLOGY Real time FEMOFLOR Real time PCR Kit FEMOFLOR 8 Real time PCR Kit FEMOFLOR 16 Content of the kit Content of the kit B C Paraffin sealed PCR mix 24 strips TS Td n ue sess Taq polymerase in buffer 4 tubes cu q In buffer w ubes z Mineral cil 4 tubes al tubas Positive control 1 tube a 0101R 2013 10 04 _ 3 R1 P802 S3 5EU Obelis European Authored Representative Genter OLE A R C ec REP B4
27. tion The PCR mix contains target specific hydrolyzing probes bearing reporter and quencher molecules While the probe is intact these molecules are close enough to provide effective quenching Once hybridized to a target sequence the probe is hydrolyzed by Taq polymerase Thereby reporter and quencher become separated and fluorescence increases proportionally to target sequence amplification The intensity of fluorescence is analyzed with a Real time PCR instrument data collection unit and the software provided The fluorescent dyes are assigned to individual types of sequences The FAM dye label is used to detect specific sequences The HEX dye label is used to detect SIC and IC see tables 1 2 3 for the reference Use of two distinguishable dyes allows detection of both PCR products simultaneously Defined tubes contain additional probe with ROX dye label Marker see tables 1 2 3 for the reference It tags the strip orientation Upon completion of run software defines actual position of the strip by means of marker position relative to the position preset by the operator If it mismatches the software suggests rearrangement of the tubes by default In accordance with the operator order can be rearranged and saved in new file The DNA Technology DT ite DTprime or DT 96 thermal cyclers are required to run the FEMOFLOR Real time PCR s Upon completion of the run software performs relative quantitative analysis of total bacterial
28. tive negative negative Negative Less than 3 0 Negative FEMOFLOR 8 Identifier Acceptable quantity of DNA in K Lg Less than 3 5 Less than 2 5 Less than 2 5 Less than 2 5 Less than 2 5 mg Qzmsgles lt O c D a S IDIS c0 QO o 3 zels Bele 3 5 3 SiS 3 S 5 45 E WY n lt 5 C UO Q 3 Cy tQ D M Ea 3 Total bacterial DNA Lactobacillus spp Gardnerella vaginalis Prevotella Less than 2 5 negative negative Less than 3 0 Negative 7 SIC FEMOFLOR 4 dentifier Acceptable quantity of DNA in K Lg Less than 3 5 Less than 2 5 Less than 2 5 1 Less than 30 negative gt 4 3 x v OlT lt gt 5 gt q 3885 Sake S nS r D Sy SIC 20 10 Storage and handling requirements 10 1 Expiry date 9 month from the date of production 10 2 All components of FEMOFLOR Real time PCR Kit must be stored at 2 8 C over the storage period 10 3 Transportation can be held by all types of roofed transport at 2 8 C over the storage period 10 4 An expired FEMOFLOR Real time PCR Kit must not be used 10 5 We strongly recommend following the instructions to get robust and reliable results 10 6 The conformity of FEMOFLOR Real
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