Influence of Design Probe and Sequence Mismatches on the Efficiency of Fluorescent RPA

Overview

Journal World J Microbiol Biotechnol

Publisher Springer

Specialties Biotechnology
Microbiology

Date 2019 Jun 13

PMID 31187258

Citations 18

Authors

Xiaoqing Liu

Qiongying Yan

Jianfei Huang

Jing Chen

Zhengyang Guo

Zhongdong Liu

Lin Cai

Risheng Li

Yan Wang

Guowu Yang

Quanxue Lan

Affiliations

Soon will be listed here.

Abstract

Recombinase polymerase amplification (RPA) is an isothermal amplification technique. Because of its short detection cycle and high specificity, it has been applied in various fields. However, the design of probe on the efficiency of RPA is not well understood and the effect of sequence mismatches of oligonucleotides on the performance of RPA is rarely discussed. In this study, we found that different primers with the same probe have a slight effect on the efficiency of fluorescent RPA, and different probes with the same amplified region have a great influence on the efficiency of fluorescent RPA. We summarized the design rules of probes suitable for fluorescent RPA by analyzing the experimental data. The rule is that the best distance between fluorescent groups in the probe is 1-2 bases, and the G content should be reduced as far as possible. In addition, we verified this rule by designing a series of probes. Furthermore, we found the base mismatches of the probe had a significant effect on RPA, which can lead to false positives and can change the amplification efficiency. However, 1-3 mismatches covering the center of the primer sequence only affect the amplification efficiency of RPA, not its specificity. And with an increase in the number of primer mismatches, the efficiency of RPA will decrease accordingly. This study suggests that the efficiency of fluorescent RPA is closely related to the probe. We recommend that when designing a fluorescent probe, one must consider the presence of closely related non-targets and specific bases.

Citing Articles

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

Zhang J, Li Z, Guo C, Guan X, Avery L, Banach D Angew Chem Int Ed Engl. 2024; 63(20):e202403123.

PMID: 38516796 PMC: 11073899. DOI: 10.1002/anie.202403123.

Efficient detection of Streptococcus pyogenes based on recombinase polymerase amplification and lateral flow strip.

Gao X, Cao Y, Gao Y, Hu J, Ji T Eur J Clin Microbiol Infect Dis. 2024; 43(4):735-745.

PMID: 38361135 DOI: 10.1007/s10096-024-04780-4.

Rapid detection of spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays.

Liu Y, Xiang J, Gao Y, Wang J, Liu L, Li R Heliyon. 2023; 9(10):e20794.

PMID: 37860527 PMC: 10582492. DOI: 10.1016/j.heliyon.2023.e20794.

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning.

Lei Z, Lian L, Zhang L, Liu C, Zhai S, Yuan X ACS Omega. 2023; 8(36):32555-32564.

PMID: 37720737 PMC: 10500685. DOI: 10.1021/acsomega.3c02929.

Rapid and sensitive detection of pathogenic in black spotted frog by RPA-LFD and fluorescent probe-based RPA.

Qiao M, Zhang L, Chang J, Li H, Li J, Wang W Fish Shellfish Immunol Rep. 2022; 3:100059.

PMID: 36419595 PMC: 9680066. DOI: 10.1016/j.fsirep.2022.100059.

References

Ishii K, Fukui M . Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Appl Environ Microbiol. 2001; 67(8):3753-5. PMC: 93085. DOI: 10.1128/AEM.67.8.3753-3755.2001. View

Piepenburg O, Williams C, Stemple D, Armes N . DNA detection using recombination proteins. PLoS Biol. 2006; 4(7):e204. PMC: 1475771. DOI: 10.1371/journal.pbio.0040204. View

Bej A, Mahbubani M, Atlas R . Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Crit Rev Biochem Mol Biol. 1991; 26(3-4):301-34. DOI: 10.3109/10409239109114071. View

Boyle B, Dallaire N, MacKay J . Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR. BMC Biotechnol. 2009; 9:75. PMC: 2741440. DOI: 10.1186/1472-6750-9-75. View

Allerberger F, Wagner M . Listeriosis: a resurgent foodborne infection. Clin Microbiol Infect. 2009; 16(1):16-23. DOI: 10.1111/j.1469-0691.2009.03109.x. View

Lutz S, Weber P, Focke M, Faltin B, Hoffmann J, Muller C . Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA). Lab Chip. 2010; 10(7):887-93. DOI: 10.1039/b921140c. View

Klungthong C, Chinnawirotpisan P, Hussem K, Phonpakobsin T, Manasatienkij W, Ajariyakhajorn C . The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR. J Clin Virol. 2010; 48(2):91-5. DOI: 10.1016/j.jcv.2010.03.012. View

Patil K, Singh P, Muniyappa K . DNA binding, coprotease, and strand exchange activities of mycobacterial RecA proteins: implications for functional diversity among RecA nucleoprotein filaments. Biochemistry. 2010; 50(2):300-11. DOI: 10.1021/bi1018013. View

Euler M, Wang Y, Nentwich O, Piepenburg O, Hufert F, Weidmann M . Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus. J Clin Virol. 2012; 54(4):308-12. DOI: 10.1016/j.jcv.2012.05.006. View

10.

Boyle D, Lehman D, Lillis L, Peterson D, Singhal M, Armes N . Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification. mBio. 2013; 4(2). PMC: 3622927. DOI: 10.1128/mBio.00135-13. View

11.

Ledeker B, De Long S . The effect of multiple primer-template mismatches on quantitative PCR accuracy and development of a multi-primer set assay for accurate quantification of pcrA gene sequence variants. J Microbiol Methods. 2013; 94(3):224-31. DOI: 10.1016/j.mimet.2013.06.013. View

12.

Abd El Wahed A, El-Deeb A, El-Tholoth M, Abd El Kader H, Ahmed A, Hassan S . A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus. PLoS One. 2013; 8(8):e71642. PMC: 3748043. DOI: 10.1371/journal.pone.0071642. View

13.

Krolov K, Frolova J, Tudoran O, Suhorutsenko J, Lehto T, Sibul H . Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples. J Mol Diagn. 2013; 16(1):127-35. DOI: 10.1016/j.jmoldx.2013.08.003. View

14.

Murinda S, Ibekwe A, Zulkaffly S, Cruz A, Park S, Razak N . Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification. Foodborne Pathog Dis. 2014; 11(7):529-36. DOI: 10.1089/fpd.2013.1663. View

15.

Jaroenram W, Owens L . Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome. J Virol Methods. 2014; 208:144-51. DOI: 10.1016/j.jviromet.2014.08.006. View

16.

Kersting S, Rausch V, Bier F, von Nickisch-Rosenegk M . Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. Mikrochim Acta. 2014; 181(13-14):1715-1723. PMC: 4167443. DOI: 10.1007/s00604-014-1198-5. View

17.

Crannell Z, Rohrman B, Richards-Kortum R . Equipment-free incubation of recombinase polymerase amplification reactions using body heat. PLoS One. 2014; 9(11):e112146. PMC: 4221156. DOI: 10.1371/journal.pone.0112146. View

18.

Daher R, Stewart G, Boissinot M, Boudreau D, Bergeron M . Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes. 2014; 29(2):116-21. DOI: 10.1016/j.mcp.2014.11.005. View

19.

Santiago-Felipe S, Tortajada-Genaro L, Morais S, Puchades R, Maquieira A . Isothermal DNA amplification strategies for duplex microorganism detection. Food Chem. 2014; 174:509-15. DOI: 10.1016/j.foodchem.2014.11.080. View

20.

Crannell Z, Rohrman B, Richards-Kortum R . Development of a quantitative recombinase polymerase amplification assay with an internal positive control. J Vis Exp. 2015; (97). PMC: 4401391. DOI: 10.3791/52620. View