RTA HIV-1 Real-Time PCR Kit
Contents
1. RTA HIV 1 Real Time PCR Kit Handbook Date of Issue 2011 04 Quantitative detection of Human Immunodeficiency Virus type 1 RNA For in vitro diagnostic use For use with the Stratagene MX3000p MX3005p and INCEPTRA Cycler 4840 9620 9640 9660 9680 Instruments For professional use only 09013025 25 tests REF 09013100 100 tests CE 0483 Table of Contents Kit Contents s22 2s2 2ecnec eecseseeeeesen le stele dee ii alla eee Ie 4 S Ola g KR An AR eR A SP Rak ek Rak k kahkaha kei e N 4 Intended Use e A NEN NEN oN NEN SN htt r naanin nooi 5 Product Use Limitations 5 Product Description nn nnn nn nnn nanan a 6 Pathogen Informiatiny 344 28 ee ee ee eee 7 Warnings and Precautions _ 8 Additional Materials Required 22 2 2 424 4242424 4 4 9 Sample Preparation 2 ee ee ee ee A AATAS ASA Rees 10 Protocol eek aa sie etre Se rom ee ar fae ver ear SN e ce ein min NASER AL n aim ers aes 11 Data Analysis Anapa tet SI SI IRITI SIS ONONO SAn E sees e eae acne 14 Performance Characteristics 16 Kit Contents Storage E Reagents 25 tests 100 tests 1 BROWN HIV 1 Reaction Mix 500 u 2x1000 yl 2 vey oyy HIV 1 Enzyme Mix 33 ul 130 u 3 BLUE HIV 1 Internal Con2. 15 Performance Characteristics Analytical Sensitivity Analytical sensitivity was analyzed by use of a dilution series of WHO standard and the cutoff value of the kit was determined by probit analysis for STRATAGENE Mx3000p and INCEPTRA Cycler 9660 Real Time PCR Systems A dilution series of a WHO International HIV 1 standard NIBSC code 97 650 was prepared to give the final concentrations of 1000 500 200 100 50 and 25 IU ml Dilutions were extracted by RTA Viral Nucleic Acid Isolation Kit Cat No 09029 according to RTA Viral Nucleic Acid Isolation Kit Handbook Starting sample volumes were 500 ul and elution volumes were 50 ul Each dilution was tested in 24 replicates for each instrument Lower limit was calculated by probit analysis done by PASW Statistics 18 program The 95 cutoff concentration of RTA HIV 1 Real Time PCR Kit is 56 IU ml for STRATAGENE and 85 IU ml for INCEPTRA Cycler INCEPTRA Cycler STRATAGENE RTA Viral Nucleic Acid Isolation Kit Starting 85 IU ml 56 IU ml sample volume 500 ul Elution volume 50 ul 16 Performance Characteristics Linear Range To determine the upper limit a dilution series of Acrometrix OptiQuant HIV 1 RNA Quantification Panel Cat no 942013 ranging from 1 x 10 IU ml to 1 x 10 IU ml were prepared Viral RNA was extracted from standards by RTA Viral Nucleic Acid Isolation Kit Cat No 09029 according to RTA Viral Nucleic Acid Isolation Kit Handbook Star
3. C 60 sec 60 C 60 sec 72 C 30 sec 72 C 30 sec Fluorescence is measured at 72 C FAM and HEX VIC JOE channels should be chosen 13 Data Analysis Following results can be interpreted gt If a signal is detected in FAM channel it is a POSITIVE result Concentration of each positive sample will be calculated by the software according to the standard curve as International Unit per milliliter IU ml Due to different starting sample volumes and elution volumes during viral DNA isolation the following formula SHOULD be used to calculate the concentration of the original clinical sample Concentration of Concentration from Software IU ml x Elution Volume ul the Original Sample IU ml Original Sample Volume ul gt If no signal is detected in FAM channel Ct240 and a signal is detected in HEX channel Ct 33 5 itis a NEGATIVE result gt If no signal is detected in FAM channel Ct240 and HEX channel Ct gt 38 NO DIAGNOSTIC INTERPRETATION can be done 14 Data Analysis Following table shows typicval Ct values of Quantification Standards for different real time PCR systems Expected Ct values INSTRUMENTS Quantification Quantification Quantification Quantification Standard 1 107 IU ml Standard 2 109 IU ml Standard 3 105 IU ml Standard 4 104 IU ml STRATAGENE 23 6042 26 7042 30 0042 33 4042 Mx3000p Mx3005p INCEPTRA Cycler 23 5042 27 0042 30 0022 33 002
4. EDTA plasma specimens were used None of the HIV 1 negative clinical specimens gave positive test result for HIV 1 RNA Diagnostic specificity of RTA HIV 1 Real Time PCR Kit is 100 All of the Internal Controls of tests gave positive result 19 Performance Characteristics Cross Reactivity potential cross reactive markers given in the table below To examine the specificity of an assay cross reactivity studies should be performed for po markers In this study the specificity of the assay was evaluated by testing 7 reference organism and 9 clinical specimens which were positive RTA HIV 1 Real Time PCR Kit do not show any cross reactivity with other ential cross reactive Organism Source Test Result Cytomegalovirus CMV Acrometrix Cat No 94 2014 Negative Human Herpes Simplex virus type 1 HSV 1 NIBSC Cat No 08 224 Negative Human Herpes Simplex virus type 2 HSV 2 NIBSC Cat No 08 226 Negative Epstein Barr Virus EBV NIBSC Cat No 08 316 Negative Hepatitis B virus HBV NIBSC Cat No 97 750 Negative Hepatitis C virus HCV NIBSC Cat No 06 100 Negative Mycobacterium tuberculosis ATCC 25177 Negative HTLV 1 3 samples Clinical specimens Negative Rous sarcoma virus Clinical specimens Negative Parvovirus B19 Clinical specimens Negative Epstein Barr Virus EBV Clinical specimens Negative Hepatitis C virus HCV 2 samples Clinical specimens Negative Cytomega
5. Inter batch precision associated with Ct values was 0 8 of variation coefficient for 10 IU ml of target RNA Precision for INCEPTRA Cycler 9660 instrument associated with Ct values was 2 03 of variation coefficient for 10 IU ml of target RNA Precision for LightCycler 2 0 instrument associated with Ct values was 0 20 of variation coefficient for 10 IU ml of target RNA And overall precision associated with Ct values was 3 15 of variation coefficient for 10 IU ml of target RNA The results on basis of Ct values are shown in the following table N Mean Std Variance Coefficient of variation 96 Deviation Intra assay 24 32 1046 543229 187 1 34 Inter assay 24 31 6471 25503 065 0 8 Inter batch 24 31 6621 25228 064 0 8 LightCycler 24 29 9063 06378 004 02 Inceptra 24 30 0813 61747 381 2 03 TOTAL 120 31 0803 97949 959 3 15 18 Performance Characteristics Genotype Detectability The performance of RTA HIV 1 Real Time PCR Kit was evaluated with SeraCare Life Sciences HIV 1 RNA Genotype Performance Panel As a result of this study RTA HIV 1 Real Time PCR Kit can detect and quantify all members of Major group of HIV 1 genotypes A B C D AE F AG G H Diagnostic Specifity HIV 1 negative clinical specimens were analyzed to determine the diagnostic specificity of RTA HIV 1 Real Time PCR Kit 62 HIV 1 RNA negative clinical serum specimens and 48 HIV 1 RNA negative clinical
6. R reaction tubes or capillaries for each sample Add 20 ul RNA of each sample negative control and quantification standards into the tubes Spin down briefly Perform the following protocol for Stratagene MX3000p MX3005p 45 C for 30 min 95 C for 10 min 1 cycle 95 C for 30 sec 63 C for 60 sec 72 C for 30 sec 45 cycles Perform the following protocol for INCEPTRA Cycler 4840 9620 9640 9660 9680 45 C for 30 min 95 C for 10 min 1 cycle 95 C for 30 sec 60 C for 60 sec 72 C for 30 sec 45 cycles Fluorescence is measured at 72 FAM and HEX channels should be chosen See the schema in the next page Refer to the Operator s Manual of the related instruments to program and analyze the results During analysis on STRATAGENE software adjust threshold fluorescence value manually by entering 1000 for FAM and 500 for HEX During analysis on INCEPTRA Cycler software adjust baseline value manually by entering 100 for both FAM and HEX 12 Protocol 1 Step Preparation of Master Mix 18 8 pl a sa Master Mix j HIV 1 Enzyme Mix m ei 12 ul lt 2 Step Addition of Samples YY Master Mix 20 ul PCR Mix gt 40 ul Sample RNA NC QS Sal 20 yl e 3 Step Programming of Thermal Cycler Program Name Cycles Program for Stratagene Program for INCEPTRA CDNA Synthesis 1 45 C 30 min 45 C 30 min Hot Start 1 95 C 10 min 95 C 10 min 95 C 30 sec 95 C 30 sec Amplification 45 63
7. ains are substantially more common in the global epidemic than the group O Outlier strains which are largely confined to Africa with sporadic cases reported elsewhere The group N non M non O strains have only been isolated in Cameroon Warnings and Precautions All clinical specimens and all resulting waste materials should be treated as potentially infectious the samples should be prepared in Bio safety Level 2 area Before and after work all surfaces should be disinfected with a freshly prepared solution of 10 bleach or antiviral agents Dispose of unused reagents waste and specimens in accordance with country or local regulations Do not pipette by mouth Do not eat drink or smoke in laboratory work areas Wear protective disposable gloves laboratory coats and eye wear when handling clinical specimens and kit reagents Wash hands thoroughly after handling specimens and test reagents Avoid contact of reagents with the skin eyes or mucous membranes If contact does occur immediately wash with large amounts of water The procedures should preferably be performed in four separated areas i e for RNA extraction PCR setup sample addition amplification to aid in preventing contamination All supplies for a particular procedure should be stored in the area where that procedure is performed and should not be moved between areas Gloves should be removed and disposed of before leaving one area to proceed to the next Lab coats sho
8. e diagnosis of infection with HIV 1 All reagents of the kit are for in vitro diagnostic use only RTA HIV 1Real Time PCR Kit is not intended for screening of blood and blood products for the presence of HIV 1 RNA or confirmation of the diagnosis of infection with HIV 1 This kit has been validated for use with human serum or human plasma collected in EDTA anticoagulant Test with other sample types may result in inaccurate results This kit has been validated for use with RTA Viral Nucleic Acid Isolation Kit Using other isolation kits may adversely affect the performance characteristics of the kit This kit has been validated for use with Stratagene Mx3000p Mx3005p instrument or INCEPTRA Cycler 4840 9620 9640 9660 9680 Using other instruments may adversely affect the performance characteristics of the kit Trustworthy results depend on proper sample collection transport storage and processing methods It is intended for professional use by properly trained personnel RTA HIV 1 Real Time PCR Kit is intended for use as an aid in the management of patients with chronic HIV 1 infection undergoing anti viral therapy to assess response to treatment in conjunction with all relevant clinical and laboratory findings The instructions in user manual should be followed strictly for optimum PCR results The expired kits should not be used Kit components from different lots should not be mixed 5 Product Description RTA HIV 1 real time PCR as
9. er free gloves Micropipettes 0 5 ul 1000 ul Sterile micropipette tips with filters Microcentrifuge tubes Vortex mixer Desktop microcentrifuge for 2 0 ml tubes and for PCR strip tubes Real Time PCR reaction tubes plates capillaries PCR workstation Sample Preparation This kit has been validated for use with human serum or human plasma collected in EDTA anticoagulant Aseptic techniques must be employed during collection to prevent the introduction of micro organisms into the patient s anatomical space and to prevent the sample from being contaminated during the process of collection All samples should be regarded as potentially infectious and standard precautions guidelines should be followed by all healt hcare workers during sample collection and handling Samples must be collected into appropriate containers before despatch to the laboratory Be careful to prevent and labora Ensure tha to check for cracks in the containers and to ensure that the lids of containers are properly tightened leakage of samples during handling and transportation This can pose infection hazards to transport ory staff the outer surfaces of the containers are not contaminated by the patients samples Store whole blood at room temperature for no longer than 4 hours Centrifuge blood within 4 hours of collection Transfer serum or plasma to a screw cap cryovial tube Transporta ion of whole blood serum or p
10. lasma must conform to country or local regulations for the transport of etiologic agents Serum or pl asma samples may be stored at 2 8 C for up to 3 days or frozen at 70 C or colder for long term storage Avoid multi ple freeze thaw cycles of specimens 10 Protocol Viral RNA Isolation RTA Viral Nucleic Acid Isolation Kit Cat No 09029 RTA Laboratories Turkey should be used for viral RNA extraction from clinical samples Please follow the manufacturer s instructions as stated in the kit manual Internal Control During RNA isolation addition of the supplied internal control IC is necessary IC allows the user to monitor RNA extraction step as well as to determine any PCR inhibition For each sample add 2 5 ul IC together with Solution RL of the Viral Nucleic Acid Isolation Kit for a 50 ul elution Depending on your final elution volume the volume of IC to be added can be calculated 0 05 ul IC 1 ul Elution Buffer There was no amplification of internal control in the tests where high positive HIV 1 samples were amplified because there was a competition between internal control template and HIV 1 RNA template for using PCR primers and other components The Ct value of internal control of a negative sample should be equal to 33 5 otherwise it denotes a problem during purification Quantification Standards For generating a standard curve to obtain accurate quantification data on the Real Time system four qua
11. lovirus CMV Clinical specimens Negative 20 Performance Characteristics Cross Contamination In this study cross contamination between samples was evaluated To do this five different runs were performed In every run 4 high positive HBV sample and 4 HIV 1 negative samples were used Samples were extracted by RTA Viral Nucleic Acid Isolation Kit according to RTA Viral Nucleic Acid Isolation Kit Handbook Starting sample volumes were 500 ul and elution volumes were 50 ul Then the kit was evaluated accordingly whether or not any cross contamination was observed No cross contamination was observed during the whole process and none of the human serum samples exhibited evidence of containing PCR inhibitors as indicated by the amplification of internal control Whole System Failure 96 HIV 1 RNA negative clinical serum specimens and 36 HIV 1 RNA negative clinical EDTA plasma specimens were spiked with WHO International HIV 1 standard NIBSC code 97 650 to give a final concentrations of 11 IU ul in the elution volume which is 3 times the 95 positive cutoff value determined by analytical sensitivity study Whole system failure rate of RTA HIV 1 Real Time PCR Kit is lt 1 96 21 RTA BR 004 Revision Date Revision No 09 02 2012 3 RTA Laboratuvarlar Biyolojik r nler la San ve Tic Ltd ti Adress Cumhuriyet Cad No 3 GEPOSB 41400 Gebze Kocaeli Phone 0262 648 5300 Fax 0262 751 0677 E mail info rtalabs co
12. m tr Web www rtalabs com tr
13. ng a singlestranded positive sense ribonucleic acid RNA genome of about 9 7 kilobases There are two strands of HIV RNA and each strand has a copy of the virus s nine genes The RNA is surrounded by a cone shaped capsid which consists of approximately 2000 copies of the p24 viral protein Surrounding the capsid is the viral envelope The viral envelope is composed of a lipid bilayer membrane formed from the cellular membraneof the host cell during budding of the newly formed virus particle Host cell proteins such as the major histocompatibility complex MHC antigens and actin remain embedded within the viral envelope along with the viral envelope protein Each envelope subunit consists of two non covalently linked membrane proteins glycoprotein gp 120 the outer envelope protein and gp41 the transmembrane protein that anchors the glycoprotein complex to the surface of the virion The envelope protein is the most variable component of HIV although gp120 itself is structurally divided into highly variable V and more constant C regions The variability of V regions may be a product of envelope functionality as has been especially well described in V3 where amino acid changes alter coreceptor use The variability of the HIV envelope also confers a uniquely complex antigenic diversity HIV 1 is divided into three quite distinct lineages the groups M N and O Again the worldwide distribution of these groups is not equal group M for Main str
14. ntification standards should be used For each standard the corresponding concentration should be defined properly to the Real Time PCR system before each run and the standard curve will be generated accordingly at the end of the reaction Work with HIV 1 Quantification Standards after preparation of clinical samples and negative control in a separate area Caps of the tubes or capillaries of Clinical Samples SHOULD be closed in that area 11 Protocol continued v v PCR Protocol Thaw all components except HIV 1 Enzyme Mix at room temperature Thaw HIV 1 Reaction Mix at 37 C for 5 min if there is a precipitate Put HIV 1 Enzyme Mix on ice Mix each component thoroughly then centrifuge briefly before use Transfer all the reagents onto ice or cooling block The final volume of Master Mix is calculated by multiplying single reaction volumes of Reaction Mix and Enzyme Mix by the total sample size The number of negative controls quantification standards and the clinical samples should be included when calculating total sample size Against possible pipetting errors addition of an extra sample to the total sample size is recommended PCR Grade Water should be used as the negative control To prepare master mix add 18 8 ul of HIV 1 Reaction Mix brown tube and 1 2 ul of HIV 1 Enzyme Mix yellow cap for each sample to the master mix tube Vortex the tube and spin down briefly in a microcentrifuge Add 20 yl of Master Mix into Real Time PC
15. say is a one step real time reverse transcription PCR assay in which RNA templates are first reverse transcribed to generate complementary cDNA strands followed by DNA polymerase mediated cDNA amplification During cDNA replication in the PCR process the internal oligonucleotide hybridizes to the template and is digested by the 5 3 endonuclease activity of the Thermus aquaticus Taq DNA polymerase as the PCR primer is extended The internal oligonucleotide is digested only if cDNA replication occurs separating the fluorescent and quencher molecules PCR products are detected within minutes by monitoring the increase in fluorescence that occurs exponentially with successive PCR amplification cycles The parameter Ct threshold cycle is defined as the fractional cycle number at which the fluorescence passes the fixed threshold A plot of the log of initial target copy number for a set of standards versus Ct is a straight line Quantification of the amount of target in unknown samples is accomplished by measuring Ct and using the standard curve to determine starting copy number RTA HIV 1 real time PCR assay utilizes external standards to gather quantitative results and includes an internal control which controls for target isolation and amplification Pathogen Information HIV is classified in the family Retroviridae subfamily Lentivirinae and genus Lentivirus The structure of HIV follows the typical pattern of the retrovirus family comprisi
16. ting sample volumes were 500 ul and elution volumes were 50 ul High concentration samples 1 x 10 1 x 10 and 1 x 10 IU ml were prepared by using calibrated in vitro transcribed RNA bearing HIV 1 quantification standard Within this range the relationship between log of target RNA and Ct values is linear Linear regression analyses comparing the Ct values versus log of target RNA were as follows For STRATAGENE Ct value 3 304 log of target RNA 43 85 with a correlation coefficient R of 0 998 For INCEPTRA Cycler Ct value 3 22 log of target RNA 44 00 with a correlation coefficient of 0 994 Upper limit is at least 1 x 10 IU ml for STRATAGENE and INCEPTRA Cycler Lower limit was calculated by probit analysis done by PASW Statistics 18 program according to the quantification results of HIV 1 Analytical Sensitivity Studies 95 lower confidence limit is 58 7 IU ml for STRATAGENE and 85 IU ml for INCEPTRA Cycler Dynamic ranges of RTA HCV Real Time PCR Kit For STRATAGENE 58 7 1 x 10 IU ml For INCEPTRA Cycler 85 1 x 10 IU ml 17 Performance Characteristics Precision For each experiment 24 replicates of 10 IU ml HIV 1 RNA 2 WHO International Standard NIBSC code 97 650 were used Intra assay precision associated with Ct values was 1 34 of variation coefficient for 10 IU ml of target RNA Inter assay precision associated with Ct values was 0 8 of variation coefficient for 10 IU ml of target RNA
17. trol 250 u 250 yu 4 RED HIV 1 Quantification Standart 1 107 IU ml 200 u 200 yu 5 RED HIV 1 Quantification Standart 2 105 IU ml 200 u 200 yu 6 RED HIV 1 Quantification Standart 3 105 IU ml 200 u 200 yu 7 RED HIV 1 Quantification Standart 4 104 IU ml 200 u 200 yu 8 PCR Grade Water 1000 ul 1000 ul All reagents of RTA HIV 1 Real Time PCR Kit should be stored at 20 C Storage at higher temperatures should be avoided e g 4 C Under these conditions kit contents should be stable through the expiration date printed on the label The reagents should not be freeze thawed more than 2 times otherwise the shelf of the kit will reduce During the working steps all reagents should be kept on ice Intended Use Product Use Limitations RTA HIV 1 Real Time PCR Kit is an in vitro nucleic acid amplification assay for quantification of Human Immunodeficiency virus type 1 HIV 1 RNA in human serum or plasma EDTA using RTA Viral Nucleic Acid Isolation Kit and Stratagene Mx3000p Mx3005p instrument or INCEPTRA Cycler 4840 9620 9640 9660 9680 instrument RTA HIV 1 Real Time PCR Kit is intended for use as an aid in the management of patients with chronic HIV 1 infection undergoing anti viral therapy to assess response to treatment in conjunction with all relevant clinical and laboratory findings RTA HIV 1 Real Time PCR Kit is not intended for screening of blood and blood products for the presence of HIV 1 RNA or confirmation of th
18. uld be specific to an area and never worn outside of that area The work should flow in one direction beginning in the extraction area moving to the PCR setup area in which PCR Master Mix is prepared then moving to the third area in which samples negative control and quantification standards are added finally moving to amplification area in which real time PCR equipment is run Warnings and Precautions continued Additional Materials Required Use all pipetting devices and instruments with care and follow the manufacturer s instructions for calibration and quality control to prevent sample contamination use new sterile aerosol barrier or positive displacement RNase free pipette tips and sterile pipettes Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of specimens or controls Store the kit away from any source of contaminating DNA or RNA especially amplified nucleic acid Do not mix reagents with different lot numbers or substitute reagents from other manufacturers Asingle type of HIV 1 RNA assay should be used for monitoring a patient If RTA HIV 1 Real Time PCR Kit substitutes another HIV 1 RNA assay both tests should be used in parallel for at least two subsequent samples Do not use a kit after its expiration date RTA Viral Nucleic Acid Isolation Kit Cat No 09029 RTA Laboratories Turkey Real Time PCR system Disposable powd
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