Reagent and method for fluorescence quantitative real-time PCR detection of RCL
The present invention provides a reagent and method for detecting a replication-competent lentivirus (RCL) by fluorescence quantitative real-time polymerase chain reaction (PCR). In particular, the present invention provides a primer and probe combination for detecting RCL, and a method for performing detection using said primer and probe; the present invention also provides a reagent kit comprising said primer and probe. The primer and probe combination of the present invention detects RCL with high amplification efficiency and good specificity, and can be used for RCL detection and RCL monitoring of clinical patient peripheral blood samples which may occur during a production process.
1. A reagent combination comprising:
(i) a first primer pair for specific amplification of vesicular stomatitis virus G protein (VSV-G) gene and a first probe, wherein, the first primer pair comprises: a first forward primer consisting of the sequence of SEQ ID NO: 1, and a first reverse primer consisting of the sequence of SEQ ID NO: 2;
wherein the first probe consists of the sequence of SEQ ID NO: 3 and a first detectable label; or
(ii) a second primer pair for specific amplification of VSV-G gene and a second probe, wherein, the second primer pair comprises: a second forward primer consisting of the sequence of SEQ ID NO: 4, and a second reverse primer consisting of the sequence of SEQ ID NO: 5;
wherein the second probe consists of the sequence of SEQ ID NO: 6 and a second detectable label; and further comprises
a third primer pair for specific amplification of a reference gene and a third probe,
wherein, the third primer pair comprises: a third forward primer consisting of the sequence of SEQ ID NO: 25, and a third reverse primer consisting of the sequence of SEQ ID NO: 26;
and, the third probe consists of the sequence of SEQ ID NO: 27 and a third detectable label.
2. The reagent combination of claim 1 , wherein the amplification efficiency of the reagent combination for detecting replication competent lentivirus (RCL) is ≥90%.
3. A detection kit comprising the reagent combination of claim 1 .
4. The detection kit of claim 3 , wherein the kit further comprises reagents for amplification.
5. The reagent combination of claim 2 , wherein the amplification efficiency of the reagent combination for detecting replication competent lentivirus (RCL) is ≥92%.
6. The reagent combination of claim 2 , wherein the amplification efficiency of the reagent combination for detecting replication competent lentivirus (RCL) is ≥95%.
7. The reagent combination of claim 1 , wherein the first, second and third detectable labels are fluorophores.