HIV-fluorescent PCR detection kits

Instruction manual
PCR-Fluorescent Quantitative Detection Kit for human immunodeficiency virus ?Principle?
In application of one-step RT-PCR and Taqman techniques, quantitatively detect RNA of immunodeficiency virus (HIV) in human serum or blood plasm.?Specification?
20 doses / kit?Kit Component?1 HIV detection fluorescent reaction liquid
320ml 2
2 Combined enzyme 9 ml 1
3 DEPC purified water 500ml 1
4 Negative serum 200ml 1
5 Quantity standard preparation(1-5 107copies/ml) 40ml 1 ?Reagents required?1 Trizol
2 Chloroform
3 75% ethanol
4 Isopropyl alcoholWe can order required reagents for you or provide purchasing contact ?Equipment Required?
The kit is applicable in PCR detection equipments of ABI7000?MJ?FTC-2000?PE5700?iCycler?Linegene and corresponding software.?Sample Collection?
2ml venous blood is collected with a disposable syringe and placed into a sterilized disposable tube. Then 0.2ml serum is isolated and checked. The sample serum can be checked at once or kept at -20?for 3 months. Sample transports in a 0 oC kettle with ice.?Test procedure?
Preparation & Application of Samples and Control Articles
Put 200ml serum sample and negative control article into a 1.5ml centrifuge tube respectively, and add 600ml Trizol and 200ml chloroform. Shake tubes upside down by hand for 15 times or vortex for 5 seconds. Transfer upper fluid to a fresh tube, add isopropyl alcohol in the same volume and mix by shaking, after 15 minutes ‚¬?centrifugalization at 13000rpm. Remove the supernatant gently after 15 minutes ‚¬?centrifugalization at 13000rpm. Add 700ml 75% ethanol, mix by shaking. Remove the liquid by placing the tube upside down on a piece of absorbant paper after 10 minutes ‚¬?centrifugalization at 13000rpm.Throw away the residual liquid from tubal wall to tube bottom tube by centrifugalization at 4000 rpm for 5 seconds. Remove the liquid completely with a transferpettor. .Dissolve the RNA with 20ml DEPC purified water.
Gradient dilution with HIV standard preparation by10?100?1000 times??PCR Amplification?
1. Mix N 30?l RT-PCR fluorescence reaction fluid with N 0.4?l enzyme equably(N: the number of reaction )
Suggestion:for RT reactions of N times,we should prepare RT reaction liquid by N+1times,to ensure there is enough reaction fluid for every reaction..
2. Place 30.4?l of the mixed liquor in a centrifuge tube, put 10?l of sample RNA, negative control, quantitation standard preparation of HIV and purified water .Then PCR amplification reaction needs to be done immediately.
Centrifuge tube is placed on quantitative PCR instrument.
The parameters are set up as follows:
45? 20min; 94? 2 min;
93? 10sec?56? 25sec?72? 30sec,cycling 40 times; conduct fluorescence detection at 56?,with the reaction system of 40l.?Results Analysis?
Quality control
For quantitative analysis, choose FAM fluorescence as the detection mode, with 5-15 cycling fluorescent signals as baseline adjustment. Principle of liminal value setting: liminal value line should be set just above the highest point of normal negative control. Copy number of the negative control should be UNDET. Coefficient correlation of standard curve =-0.98. Otherwise, the test is invalid
Result assessing
According to the working curve, equipment can automatically show sample ‚¬ quantitative value. The indication of ‚¬ NDET ‚¬?means the sample is negative. If Ct value is between 38~40, detect it once more. If still between 38~40, it can be assessed negative.?Transportation?
Transport in a tightly sealed kettle with ice or foam box with ice.?Storage & Duration?
6 month storage at -20 ?,away from light. Freezing and thawing PCR reaction mixture should no more than 3 times.?Warnings and Precautions?
Please read this instruction carefully before detection
1.The whole detection procedures should be strictly conducted in 3 areas: preparation area for PCR reaction system; sample preparation and application area; area for PCR amplification?fluorescence detection and result analysis. The instrument, facility, materials, and working clothes should be separately prepared for every area. Clean and sterilize the benchboard right after the detection. .
2. Use disposable gloves not containing fluorescent material (frequent replacement), special disposable centrifuge tube, auto-unloading transferpettor, and tip with filter.
3. Superclean bench (underpressure typed) or antifouling cover for preparation of reagent and sample to prevent environmental pollution.
4. Set negative and positive controls for every detection.
5. Operators should have specific training, certain experience and operating skills.
6. Frequent sterilization with 10% hypochloric acid, or 70% alcohol, or UV-lamp for benchboard transferpettor, centrifugal machine, PCR amplification equipment, etc.
7. The used tip, centrifuge tube, sample