-1 Viral Load Real Time (RG) Real Time RT-PCR type 1 RNA quantification assay MSP Reg. pending Valdense 3616. 11700. Montevideo. Uruguay. phone (598) 2 336 83 01. Fax (598) 2 336 71 60. Info@atgen.com.uy www.atgen.com.uy Page 1 of 13
Only for research. The purchase of this product does not provide a license to carry out patented applications. Kit Utility ATGen -1 Viral Load Real Time (RG) is an specific kit for the detection and quantification of Human Immunodeficiency Virus Type 1 genomic RNA. The genome number of copies are directly related to the number of free viral particles present in the sample, thus when using plasma samples as a source for viral RNA, this kit allows Viral Load determination. The system is optimized for Rotor-Gene TM 3000 and Rotor-Gene TM 6000 (Corbett Research) real time platforms. Principle of the Assay -1 Viral Load Real Time (RG) is a nucleic acid amplification assay ready to be used with viral RNA purified samples. The detection and quantification are achieved by the amplification of a short conserved genome region. Target RNA is retrotranscripted and the resultant copy-dna is amplified by polymerase chain reaction (PCR) while the fluorescence detection is monitored in each amplification step. A broad range standard curve (provided) is used to compute the number of RNA copies which are present in the samples. In order to detect false negatives due to errors in RNA purification or PCR inhibition, an homologous internal control is incorporated to each reaction. Introduction: Human Immunodeficiency Virus Type 1 The Human Immunodeficiency Virus () is the etiological agent of the Acquired Immune Deficiency Syndrome (AIDS), one of the major sexually transmitted diseases. The infection may also be acquired by exposition to infected blood or its derivatives and by vertical transmission from mother to child via placenta or at birth. Within a week after exposition, infected individuals normally develop flu like or mononucleosis like symptoms with high viral load levels in peripheral blood. In this acute stage of infection, levels of more than ten million copies/ml of RNA could be easily reached. A decline of free virus in blood is expected in most individuals after the stimulation of the immune system, usually 4 to 6 weeks after the first symptoms. This seroconversion indicates the beginning of the chronic stage of the infection which can be clinically stable and asymptomatic for years. The chronic stage is characterized by the progressive decline of the T CD4+ lymphocyte number, leading to severe immunodeficiency and opportunistic infections, cancer and other associated diseases and finally to death. Although viral levels in plasma may be stable at this stage, the virus remains replicative. Page 2 of 13
High levels of virus in peripheral blood due to uncontrolled replication activity is directly associated with immunodeficiency development due to the T CD4+ lymphocyte clearance from blood. Furthermore, the reduction of virus in plasma due to antiretroviral therapy treatment is associated with a diminished risk of AIDS disease. Levels of free virus in peripheral blood could be measured by direct virus detection through p24 antigen, by in vitro culture in specialized laboratories or more recently by measuring the viral ARN copy number using Nucleic Acid Testing technologies as the one provided with this kit. Kit presentation The -1 Viral Load Real Time (RG) provides all the reagents needed for the real time RT-CR assay as well as for determining the number of viral RNA copies in RNA purified samples. Box 1-1 MIX, Reaction mix (4 tubes, pink cap) for up to 20 reactions: 12 samples, one standard curve, one positive control and one negative control. -1 NTC, Negative Control (1 tube, white cap) Color Label Color Label MIX MIX MIX MIX -1 NTC Page 3 of 13
Box 2-1 STD, standards for viral RNA quantification (6 tubes, red cap) -1 Carrier-CI, carrier RNA with viral RNA internal control (1 tube, blue cap) CI+, positive control for yellow channel (1 tube, yellow cap). Color Label Color Label STD1 STD1 STD3-1 Carrier-CI STD4 STD5 STD6 CI+ Necessary materials not provided with the test Rotor-Gene TM Real Time thermocycler (Corbett). Biohazard disposable container Gloves and robe. Suitable pippetes. RNase free, aerosol barrier pippete tips. RNase free PCR tubes. Vortex. RNA purification system. QIAamp Viral RNA Mini Kit (Qiagen) or similar is recommended for plasma and culture supernatant samples. Precautions 1. Only for in Vitro use. 2. All samples, reagents and controls should be considered as potentially infectious. 3. Do not use this kit after the expiration date indicated on the package. Page 4 of 13
Storage and Stability The kit must be stored at -20ºC in order to assure its optimal performance through the expiration date indicated on the package. The kit allows up to 3 defrosting cycles. In case additional defrosting would be needed, to aliquot the reaction mix in the first defrosting is recommended. Specimen Characteristics: Viral RNA copies purified using highly reproducible methods that assure both its quality and stability. Plasma from infected individuals and culture supernatants are the most common source of virus. The use of an RNA purification column based commercial systems is highly recommended. An efficient RNA extraction from samples with low viral titers requires the use of a carrier RNA during the purification process. The addition of an Internal Control RNA provides an extraction quality control for each test sample. The kit s mix of carrier RNA and internal control RNA (Carrier+CI) provides both solutions. In order to assure reproducible levels of extraction of low viral samples and to control purification procedures and PCR inhibition, use the Carrier+CI during the RNA extraction, as indicated in the Protocol section Protocol 1. Internal-Control During the lysis and ribonuclease inactivation step of the RNA purification, defrost and then add 10 µl of Carrier+IC per test sample. Homogenize by vortexing and continue with the purification as indicated in the protocol of the RNA purification system you are selected to use. If you are using already purified RNA samples, 2 µl of Carrier+CI may be added to the PCR mix in order to avoid PCR inhibition. 2. Amplification Mix preparation: 2.1 Completely defrost the MIX avoiding direct light and high temperatures. 2.2 Homogenize by vortexing during 15 seconds and spin briefly. 2.3 Pipette 15 µl of MIX in each PCR tube. 2.4 Add 10 µl of sample to each PCR tube and homogenize by pippeting. 2.5 Set up the assay as shown in Chart 1. Page 5 of 13
Chart 1 PCR tube # Sample Description 1-1 NTC Negative Control 2-1 STD1 Standard 1 (1x10 1 copies/ul) 3-1 STD2 Standard 2 (1x10 2 copies/ul) 4-1 STD3 Standard 2 (1x10 3 copies/ul) 5-1 STD4 Standard 2 (1x10 4 copies/ul) 6-1 STD5 Standard 2 (1x10 5 copies/ul) 7-1 STD6 Standard 2 (1x10 6 copies/ul) 8 PC+ Positive Control for Yellow channel 9 Test sample #1 Viral RNA test sample 10 Test sample #2 Viral RNA test sample 11 12 -- 13 14 15 16 It is advisable to keep cold the RNA samples during manipulation. The use of refrigerated tube racks is practical for this purpose. 3. Programming the instrument: Page 6 of 13
Place the PCR tubes in the rotor as indicated in Chart-1 If an Template is already stored go to step 2.2. If it is the first time you program the instrument, create a new Template for the assay: Start running the program and select Empty Run. Select New and Next. In the displayed window select tube type and click Next. In the displayed window select 25 µl as reaction volume and click Next. Select Edit Profile and set the thermocycling and acquisition protocol as follows: Temp. C Time Acquiring Hold 42 30 No Hold 2 95 10 No 95 15 sec No 45 cycles 60 20 sec Yellow Green The Edit Profile screen will show the thermocycling profile (see next figure). Check that all the cycling steps were correctly entered and that the acquisition channels were correctly selected. Then click OK. Page 7 of 13
1. Select Calibrate and in the new window Calibrate acquiring. Select Perform calibration before 1rst acquisition and click OK. In the next screen select Save Template using the name -1 Quant. After saving the template select Start Run and follow the wizard steps. Page 8 of 13
2. Instrument programming from a previous template. Start running the Rotor Gene software; in the New Run screen, select the -1 Quan template and then click New. 3. Select 36 samples rotor and then click Next. 4. Enter 25 µl as reaction volume, and then click Next. Page 9 of 13
5. In the next screen, the template cycling settings will be displayed as entered in section 2.1.E. Select Start run. 1. Select Calibrate and in the next screen, Calibrate acquiring. Select the option: perform calibration before 1rst acquisition and click Close. 2. Save the file with a selected name for the present run and click Accept. 3. Fill the sample chart with the corresponding data as shown in Table 1 and click Finish to close the window and ending the instrument programming. Page 10 of 13
Data Analysis: 1. Once the run is completed select Analysis. Then choose Quantification and select Cycling A. Green and Cycling A. Yellow. Go through the following steps in each channel analysis: a. Select Dynamic Tube and Slope Correct b. Select Outlier Removal NTC threshold = 5% c. Enter threshold = 0,03 d. Enter standard curve coefficient R 2 = 0,9 e. Enter amplification efficiency = 0,90 2. Validation Negative Control (yellow and green channel) The absence of an amplification curve for the negative controls in the yellow and green channels validates the experiment indicating there was no RNA/DNA contamination during manipulation. The presence of an amplification curve invalidates the analysis. Positive Control (yellow channel) An amplification curve with Ct between 30 and 35 should be obtained in the positive control tube position (yellow channel). This indicates amplification of the internal control. The absence of an amplification curve or a higher Ct invalidates the experiment. Standard Curve (green channel) The standard curve is constituted by 6 different titles with an R 2 0.98 coefficient and a 3.3-3.5 slope. The amplification efficiency, calculated with the standard curve, should be 92%. Deviations from these parameters affect the quantification accuracy rendering erroneous results. Test sample internal control (yellow channel) Page 11 of 13
The presence of amplification curves with Cts =30-35 in the test samples indicate efficient RNA purification. Internal control amplification can be inhibited in test samples with a high viral RNA copy number (green channel). In these cases the experiment remains valid despite Ct values greater than 35 or if there is no amplification curve in the yellow channel. For a given sample, the absence of amplification curve in both the yellow and green channels invalidates the analysis of that sample, indicating poor RNA purification procedures or PCR inhibition. Bibliography: 1. Semple M, Loveday C, Weller I, Tedder R: Direct Measurement of Viraemia in Patients Infected With -1 and Its Relationship to Disease Progression and Zidovudine Therapy, J Med Vir 1991; 35:38-45. 2. Lu W, Andrieu JM: Early Identification of Human Immunodeficiency Virus-Infected Asymptomatic Subjects Susceptible to Zidovudine by Quantitative Viral Coculture and Reverse Transcription-Linked Polymerase Chain Reaction, J Inf Dis 1993; 167:1014-1020 3. Piatak M, Saag MS, Lifson JD, et al: High Levels of -1 in Plasma During All Stages of Infection Determined by Competitive PCR, Science 1993; 259:1749-1754. 4. Mellors JW, Kingsley LA, Kokka RP et al: Quantitation of -1 RNA in plasma predicts outcome after seroconversion. Ann. Intern. Med. 1995; 122:573-579. Notes: Page 12 of 13
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