Duplex Detection of VibrioCholerae and VibrioParahaemolyticus by Real-time Recombinase Polymerase Amplification.
Lei LiaoWen ShiChao MaWen Lian TangQi Lan QianYu WangJiao Jiao LiJin Yang ShenJing JiMing JinSong Gao
1
Citation
0
Reference
10
Related Paper
Citation Trend
Keywords:
Recombinase Polymerase Amplification
Duplex (building)
Peptide nucleic acid
Molecular diagnostics
Cite
Citations (0)
Abstract Background Milk vetch dwarf virus (MDV) is an important ssDNA virus which causes yellowing, stunting and leaf rolling symptoms on legumes. In China, the virus causes great economic losses and has recently been found to infect tobacco. The expansion of its host range and its ability to spread rapidly has given rise to the urgent need for a sensitive, specific and rapid diagnostic assay that can assist in effective disease control. Methods Assays based on the polymerase chain reaction combined with lateral flow strip detection (PCR-LFS) and recombinase polymerase amplification combined with LFS (RPA-LFS) were developed targeting the coat protein (CP) gene of MDV. Results The PCR and RPA assays could detect respectively 10 3 copies or 10 1 copies of MDV by agarose gel electrophoresis. The PCR-LFS and RPA-LFS assays developed could both detect as few as 10 1 copies per reaction at 37 °C. Both methods could detect MDV in crude leaf extracts. Conclusions The RPA-LFS assay developed is a rapid, sensitive and specific method for detecting MDV, which is convenient and has great potential for use in the field.
Recombinase Polymerase Amplification
Agarose gel electrophoresis
Cite
Citations (22)
Multiple displacement amplification
Hot start PCR
Primer dimer
Applications of PCR
Recombinase Polymerase Amplification
Ligase chain reaction
Cite
Citations (16)
Recombinase Polymerase Amplification
Clostridium perfringens
Cite
Citations (0)
재조합효소-중합효소 증폭법(RPA)은 등온에서 매우 빠른 반응속도를 보이는 유전자 증폭법으로 근래 여러 병원체의 검출법에 폭넓게 적용되고 있다. 본 연구는 kSBV의 빠른 검색을 위하여 kSBV 특이 실시간 재조합효소-중합효소 증폭법을 새로이 개발하였으며, 특이 cDNA로부터 2분 46초만에 kSBV특이 유전자의 존재를 확인할 수 있었다. 또한, 이를 바탕으로 목적 유전자의 정량이 가능한, 보편적 응용이 가능한 정량 실시간 재조합효소-중합효소 증폭법(quantitative real-time RPA)을 제안하였으며, 이 정량법이 현장시료에서 유용함을 입증하였다. 본 실험법은 꿀벌 질병체의 정량 검출 뿐 아니라, 일반 병원체의 정량 검출에서도 응용되기를 기대한다.
Recombinase Polymerase Amplification
Cite
Citations (0)
Polymerase chain reaction (PCR) is a molecular biology technique used to multiply certain deoxyribonucleic acid (DNA) fragments. It is a common and indispensable technique that has been applied in many areas, especially in clinical laboratories. The third generation of polymerase chain reaction, droplet digital polymerase chain reaction (ddPCR), is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify DNA. Droplet digital polymerase chain reaction is now widely used in low-abundance nucleic acid detection and is useful in diagnosis of infectious diseases. Here, we summarized the potential advantages of droplet digital polymerase chain reaction in clinical diagnosis of infectious diseases, including viral diseases, bacterial diseases and parasite infections, concluded that ddPCR provides a more sensitive, accurate, and reproducible detection of low-abundance pathogens and may be a better choice than quantitative polymerase chain reaction for clinical applications in the future.
Applications of PCR
Recombinase Polymerase Amplification
Molecular diagnostics
Cite
Citations (202)
Multiple displacement amplification
Denaturation (fissile materials)
Recombinase Polymerase Amplification
Hot start PCR
Taq polymerase
Cite
Citations (8)
Recombinase Polymerase Amplification
Cite
Citations (10)
TaqMan
Hot start PCR
Taq polymerase
Primer dimer
Applications of PCR
Multiple displacement amplification
Cite
Citations (27)
Objective To increase the sensitivity of quantitative PCR using DNA polymerase in combination with aptamer 6-10.Methods The quantitative PCR was performed using DNA polymerase mixed with aptamer 6-10 or specific antibody.The low detection limit of each modified method was compared with that of the conventional method.Results The quantitative PCR using aptamer 6-10(200 nmol/L) or specific antibody increased the low detection limit of human death receptor 5(DR5) plasmid from 105 copies/μl to 103 copies/μl.Melting curves showed that each method had minimal nonspecific amplification when the concentration of DR5 plasmid was 105 copies/μl;however,when the concentration of DR5 plasmid was 103 copies/μl,the conventional method had nonspecific amplification,whereas the method using aptamer 6-10 had minimal nonspecific amplification.Conclusions Aptamer 6-10 of DNA polymerase can increase the sensitivity of quantitative PCR.
Aptamer
Multiple displacement amplification
Primer dimer
Recombinase Polymerase Amplification
Cite
Citations (0)