US 12,467,041 B2
Pfu DNA polymerase mutants with reverse transcriptase activity and their applications
Lixin Ma, Hubei (CN); Wanping Chen, Hubei (CN); Rui Han, Hubei (CN); Fei Wang, Hubei (CN); Nianzu He, Hubei (CN); and Jiakai Cui, Hubei (CN)
Assigned to HUBEI UNIVERSITY, Wuhan (CN)
Filed by HUBEI UNIVERSITY, Hubei (CN)
Filed on Jan. 9, 2025, as Appl. No. 19/014,260.
Application 19/014,260 is a continuation of application No. PCT/CN2024/111090, filed on Aug. 9, 2024.
Claims priority of application No. 202311718419.6 (CN), filed on Dec. 12, 2023.
Prior Publication US 2025/0188430 A1, Jun. 12, 2025
Int. Cl. C12N 9/12 (2006.01); C12Q 1/686 (2018.01)
CPC C12N 9/1252 (2013.01) [C12Q 1/686 (2013.01); C12Y 207/07007 (2013.01)] 2 Claims
 
1. A method for detecting ribonucleic acid (RNA), comprising the following steps:
using RNA as a template,
performing reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) using a Pyrococcus furiosus deoxyribonucleic acid (Pfu DNA) polymerase mutant; and
qualitatively or quantitatively analyzing the RNA through fluorescence results,
wherein the Pfu DNA polymerase mutant is selected from:
K467R/F588L/W769R mutant, wherein the 467th, 588th, and 769th positions of the amino acid sequence shown in SEQ ID NO: 1 to arginine, leucine, and arginine, respectively;
R382H/Y385H/V390I mutant, wherein the 382nd, 385th, and 390th positions of the amino acid sequence shown in SEQ ID NO: 1 to histidine, histidine, and isoleucine, respectively;
I38L/R97M mutant, wherein the 38th and 97th positions of the amino acid sequence shown in SEQ ID NO: 1 to leucine and methionine, respectively;
E665K/E735K mutant, wherein the 665th and 735th positions of the amino acid sequence as shown in SEQ ID NO: 1 into lysine; and
K118I/N713V mutant, wherein the 118th and 713th positions of the amino acid sequence shown in SEQ ID NO: 1 to isoleucine and valine, respectively.