Replication-competent Lentivirus (RCL) Detection Kit _41311ES

Replication-competent Lentivirus (RCL) Detection Kit is used to quantitatively detect replicating lentiviruses that may occur in a variety of cell products associated with lentiviral vectors potential risks.

This kit designs specific primers for the VSV-G gene sequence of lentiviral envelope proteins. And it adopts taqman   fluorescent probe and the polymerase chain reaction (PCR) method, which has 1 copies/μL level detection limit and can specifically and quickly detect the replication-competent lentivirus risk. The kit needs to be used together with the the Residual DNA Sample Preparation Kit (Cat# 18461ES).

 

Components 

Components No.	Name	41311ES50	41311ES60
41311-A	RCL qPCR Mix	0.75 mL	1.5 mL
41311-B	RCL Primer&Probe Mix	200  μL	400  μL
41311-C	DNA Dilution Buffer	2×1.8 mL	4×1.8 mL
41311-D	RCL DNA Control（5×10E8 copies/μL)）	25 μL	50 μL
41311-E	IC*	50 μL	100 μL

* IC： Internal control.

Storage

This product should be stored at -25~-15℃ for 2 years.

Both 41311-A and 41311-B should be stored protected from light.

Applicable instrument models

Include but not limited to: Bio-Rad: CFX96 Optic Module; Thermo Scientific: ABI 7500; ABI Quant Studio 5; ABI Step OnePlus.

 

Instructions

1. RCL DNA Standard dilution and Standard curve preparation

The RCL DNA Control was gradient diluted using the DNA Dilution Buffer provided in the kit* , and the dilution concentration is 5×10E7 copies/μL, 5×10E6 copies/μL, 5×10E5 copies/μL, 5×10E4 copies/μL, 5×10E3 copies/μL, 5×10E2 copies/μL, 5×10E1 copies/μL.

See detailed instructions below:

1) Thaw the RCL DNA control and DNA dilution buffer on ice. After completely thawed, vortex gently to mix, and centrifuge at low speed for 10 secs.

2) Take out seven clean 1.5 mL tubes, marked with Std0, Std1, Std2, Std3, Std4, Std5, Std6.

3) Add 90  μL of DNA dilution buffer and 10  μL of RCL DNA Control to the 1.5 mL microfuge tube labeled Std0, namely   dilute to 5×10E7 copies/μL. Mix and then centrifuge for 10 secs. Subpackage the diluted DNA standard and it can be stored in the short term (no more than 3 months) at -25~-15℃** .Please avoid repeated freeze-thaw.

4) Add 90  μL of DNA dilution buffer into other tubes*** , then follow the below procedure for the serial dilutions**** .

 

Tube	Dilution Ratio	Standard concentration
Std1	10 μL Std0 + 90 μL DNA Dilution Buffer	5×10E6 copies/μL
Std2	10  μL Std1 + 90  μL DNA Dilution Buffer	5×10E5 copies/μL
Std3	10  μL Std2 + 90  μL DNA Dilution Buffer	5×10E4 copies/μL
Std4	10 μL Std3 + 90 μL DNA Dilution Buffer	5×10E3 copies/μL
Std5	10  μL Std4 + 90  μL DNA Dilution Buffer	5×10E2 copies/μL
Std6	10  μL Std5 + 90  μL DNA Dilution Buffer	5×10E1 copies/μL

Table1 Standard gradient dilution

*Three replicate wells are required for each concentration.The detection range is 5×10E1 copies/μL~5×10E6 copies/μL and this range can be expanded if required.

** To reduce the number of repeat freeze-thaw and avoid contamination, it is recommended to store the DNA control in aliquots at -25~-15℃ for the first time.

*** Once thawed, DNA dilution buffer could be stored at 2-8°C for 7 days, if not used for a long time, please store at -25~-15℃ .

**** Make sure the template is completely mixed, gently shake the mixture for 15 secs to 1 min for each gradient dilution.

2. Extraction Recovery Control (ERC) preparation

Set the concentration of RCL DNA in ERC as needed (the ERC sample was prepared with 5×10E4  copies RCL DNA as an example), as follows:

1) Add 100  μL test sample into a clean 1.5 mL tube, then add 10  μL 5×10E3 copies/μL RCL DNA Standard (Std4) and mix well, marked as ERC.

2)  Perform the DNA extraction of ERC sample together with the test samples to prepare the purified ERC sample.

3. Negative Control Solution (NCS) preparation

Set the negative control in the experiment, the specific operation steps are as follows:

1) Add 100  μL sample matrix (or DNA dilution buffer) into a clean 1.5 mL tube, then marked as NCS.

2) Perform the DNA extraction of NCS sample together with the test samples to prepare the purified NCS sample.

4. No Template Control (NTC) preparation

Set the no template control in the experiment, the specific operation steps are as follows:

1) NTC requires no sample pretreatment, and can be configured at the stage of qPCR detection of residual DNA content.

2) The NTC sample in each tube or well is 20  μL Mix (i.e. 15  μL RCL qPCR Mix + 4  μL RCL Primer&probe Mix + 1 μL IC) + 10  μL DNA Dilution Buffer. It is recommended to configure three replicate wells.

5. PCR reaction system

 

Component	Volume(μL)
RCL qPCR Mix*	15
RCL Primer&Probe Mix	4
IC	1
DNA Template	10
Total volume**	30

 

Table2 Reaction system

* Calculate the total PCR reaction volume by the number of reactions: qPCR Mix =(the number of reactions+2) × (15+4+1) μL (including the losses of two reaction wells). More than three replicates for each sample are recommended in the experiment.

** After capping the tube or sealing the plate, centrifuge the reaction tube or plate at low speed for 10 secs. After sufficient shaking and mixing for 5 secs, repeat centrifuge to collect the liquid from the lid or wall to the bottom. Avoid bubbles during operation.

See below table for the recommended Plate setup:

1

2

3

4

5

6

7

8

9

10

11

12

A

NTC

TS 1

TS 1

TS 1

Std 1

Std 1

Std 1

B

NTC

TS 2

TS 2

TS 2

Std 2

Std 2

Std 2

C

NTC

TS 3

TS 3

TS 3

Std 3

Std 3

Std 3

D

Std 4

Std 4

Std 4

E

NCS

ERC 1

ERC 1

ERC 1

Std 5

Std 5

Std 5

F

NCS

ERC 2

ERC 2

ERC 2

Std 6

Std 6

Std 6

G

NCS

ERC 3

ERC 3

ERC 3

H

Table3 Computer-on reference board

The plate layout includes: 6 Std (the standard curve of 6 standard concentrations), 1 NTC (no template control), 1 NCS (negative control solution), 3 TS (test samples), 3 ERC (extraction recovery control). Three replicate wells for each sample.

6. Setup guidelines for a PCR Instrument 

The following instructions apply only to Thermo ABI 7500 qPCR instrument (Software version 2.0). If you use a different instrument, refer to the applicable instrument guide for setup guidelines.

1) Generate a new experiment, choose the template of absolute quantification or user-defined.

2) Create 1 detection probe, named "RCL-DNA", select reporter fluorophore as "FAM" and quench fluorophore as "none"; create 1 more detection probe, name "IC" and select the reporter fluorophore as "CY5" and quenching fluorophore as "none". The reference fluorescence is ROX" (the reference fluorescence can be based on the instrument model, etc., select whether you need to add it).

3) In the ‘Samples’ pane, add all the samples information in turn. Then select the wells, choose the target and the samples correspondingly. Set the task of RCL DNA standard as standard, and assign the values 5000000, 500000, 50000, 5000, 500, 50 (the unit of DNA concentration in each well is copies/μL) in the Quantity column, and name the  wells Std 1, Std 2, Std 3, Std 4, Std 5, Std 6, correspondingly. Set the task of NTC as NTC. Set the NCS, TS, and ERC as Unknown, and named them according to the above Plate layout correspondingly. Then click next.

4) Set the amplification program: set the reaction volume as 30 μL.

Cycle Step	Temperature(℃)	Time	Cycles
Contaminated digestion	37℃	5 min	1
Initial denaturation	95℃	5 min	1
Denaturation	95℃	15 sec	45
Annealing/Extension（Fluorescence collection）	60℃	30 sec

Table4 Amplification procedure

7. Analysis of qPCR results

1) The system will automatically give the Threshold In the Amplification Plot panel of Analysis. The Threshold given by the system is too close to the baseline sometimes, resulting in a large difference in Ct between replicate wells. You can manually adjust the Threshold to an appropriate position and click Analyze. Then you can initially check whether the amplification curve is normal in Multicomponent Plot.

2) In the Result Analysis tab, review the Standard Curve plot. Verify the values for the R2, Efficiency, Slope and Y-intercept. For a normal standard curve, R²>0.99, 90%≤Eff%≤110%, -3.6≤Slope≤-3.1.

3) In the ‘View well table’ pane in Analysis, the concentrations of each samples are showed in Quantity, the unit is copies/μL, the units can be convert in the assay report.

4) The parameter settings of the result analysis need to be based on the specific model and the software version used, and can generally be automatically interpreted by the instrument.

5) Calculate the spike recovery rate based on the test results of the sample TS to be measured and the sample spike recovery ERC, the recovery rate of spikes is required to be between 50%~150%.Spiked recovery rate meter formula:

Recovery (%) = {Sample spiked assay (eg.copies/μL) - Sample assay (eg.copies/μL)} x Elution volume (μL) / Theoretical value of DNA addition amount (eg.copies) x 100%。

6) The Ct value of the negative control NCS should be greater than the mean of the lowest concentration Ct of the standard.

7) Template free control NTC should be Undetermined or Ct value ≥38.

Notes

1. This product is for research use only.
2. Please operate with lab coats and disposable gloves, foryour safety.

3.Please read this manual carefully before using this reagent, and the experiment should be standardized, including sample handling, reaction system preparation and sample addition.

4.Ensure that each component is fully vortexized and centrifuged at low speed before use.

 

Manual

41311