Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood
2019; Elsevier BV; Volume: 21; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2019.02.004
ISSN1943-7811
AutoresMindy Leelawong, Nicholas M. Adams, W. Gabella, David W. Wright, Frederick R. Haselton,
Tópico(s)Mosquito-borne diseases and control
ResumoMonitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. The probes feature two strategic design elements: locked nucleic acids to enhance specificity and the reporter dyes Cy5 and TEX615, which have less optical overlap with the blood absorbance spectra than other commonly used dyes. Probe performance was validated on a traditional laboratory-based instrument and then further tested on a field-deployable Adaptive PCR instrument to develop a point-of-care platform appropriate for use in malaria settings. The probes discriminated between wild-type Plasmodium falciparum and the chloroquine-resistant CRT PF3D7_0709000:c.227A>C (p.Lys76Thr) mutant in the presence of 2% blood. Additionally, in allelic discrimination plots with the new probes, samples clustered more closely to their respective axes compared with samples using minor groove binder probes with 6-FAM and VIC reporter dyes. Our strategy greatly simplifies single-nucleotide polymorphism detection and provides a more accessible alternative for antimalarial resistance surveillance in the field. Monitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. The probes feature two strategic design elements: locked nucleic acids to enhance specificity and the reporter dyes Cy5 and TEX615, which have less optical overlap with the blood absorbance spectra than other commonly used dyes. Probe performance was validated on a traditional laboratory-based instrument and then further tested on a field-deployable Adaptive PCR instrument to develop a point-of-care platform appropriate for use in malaria settings. The probes discriminated between wild-type Plasmodium falciparum and the chloroquine-resistant CRT PF3D7_0709000:c.227A>C (p.Lys76Thr) mutant in the presence of 2% blood. Additionally, in allelic discrimination plots with the new probes, samples clustered more closely to their respective axes compared with samples using minor groove binder probes with 6-FAM and VIC reporter dyes. Our strategy greatly simplifies single-nucleotide polymorphism detection and provides a more accessible alternative for antimalarial resistance surveillance in the field. CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2019 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2019 CME Program in Molecular Diagnostics") for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The drugs used to treat malaria have followed a pattern typical for many other antimicrobials. Initially, the drug works well, but over time, it becomes less effective through complex mechanisms that lead to the development of drug resistance. This paradigm was observed for chloroquine starting in the 1950s, then later with sulfadoxine-pyrimethamine in the following decade (reviewed by Wongsrichanalai et al1Wongsrichanalai C. Pickard A.L. Wernsdorfer W.H. Meshnick S.R. Epidemiology of drug-resistant malaria.Lancet Infect Dis. 2002; 2: 209-218Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). Indications of a similar pattern are being observed with the current first-line treatment for Plasmodium falciparum malaria.2Noedl H. Se Y. Schaecher K. Smith B.L. Socheat D. Fukuda M.M. Evidence of artemisinin-resistant malaria in western Cambodia.N Engl J Med. 2008; 359: 2619-2620Crossref PubMed Scopus (1272) Google Scholar, 3Dondorp A.M. Nosten F. Yi P. Das D. Phyo A.P. Tarning J. Lwin K.M. Ariey F. Hanpithakpong W. Lee S.J. Ringwald P. Silamut K. Imwong M. Chotivanich K. Lim P. Herdman T. An S.S. Yeung S. Singhasivanon P. Day N.P. Lindegardh N. Socheat D. White N.J. Artemisinin resistance in Plasmodium falciparum malaria.N Engl J Med. 2009; 361: 455-467Crossref PubMed Scopus (2497) Google Scholar, 4Ashley E.A. Dhorda M. Fairhurst R.M. Amaratunga C. Lim P. Suon S. et al.Tracking resistance to artemisinin C: spread of artemisinin resistance in Plasmodium falciparum malaria.N Engl J Med. 2014; 371: 411-423Crossref PubMed Scopus (1437) Google Scholar, 5Phyo A.P. Nkhoma S. Stepniewska K. Ashley E.A. Nair S. McGready R. ler Moo C. Al-Saai S. Dondorp A.M. Lwin K.M. Singhasivanon P. Day N.P. White N.J. Anderson T.J. Nosten F. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study.Lancet. 2012; 379: 1960-1966Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 6Thanh N.V. Thuy-Nhien N. Tuyen N.T. Tong N.T. Nha-Ca N.T. Dong L.T. Quang H.H. Farrar J. Thwaites G. White N.J. Wolbers M. Hien T.T. Rapid decline in the susceptibility of Plasmodium falciparum to dihydroartemisinin-piperaquine in the south of Vietnam.Malar J. 2017; 16: 27Crossref PubMed Scopus (112) Google Scholar, 7Wang Z. Shrestha S. Li X. Miao J. Yuan L. Cabrera M. Grube C. Yang Z. Cui L. Prevalence of K13-propeller polymorphisms in Plasmodium falciparum from China-Myanmar border in 2007-2012.Malar J. 2015; 14: 168Crossref PubMed Scopus (66) Google Scholar, 8Thriemer K. Hong N.V. Rosanas-Urgell A. Phuc B.Q. Ha do M. Pockele E. Guetens P. Van N.V. Duong T.T. Amambua-Ngwa A. D'Alessandro U. Erhart A. Delayed parasite clearance after treatment with dihydroartemisinin-piperaquine in Plasmodium falciparum malaria patients in central Vietnam.Antimicrobial Agents Chemother. 2014; 58: 7049-7055Crossref PubMed Scopus (70) Google Scholar, 9Ye R. Hu D. Zhang Y. Huang Y. Sun X. Wang J. Chen X. Zhou H. Zhang D. Mungthin M. Pan W. Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border.Sci Rep. 2016; 6: 20100Crossref PubMed Scopus (50) Google Scholar, 10Boulle M. Witkowski B. Duru V. Sriprawat K. Nair S.K. McDew-White M. Anderson T.J. Phyo A.P. Menard D. Nosten F. Artemisinin-resistant Plasmodium falciparum K13 mutant alleles, Thailand-Myanmar border.Emerg Infect Dis. 2016; 22: 1503-1505Crossref PubMed Scopus (27) Google Scholar, 11Huang F. Takala-Harrison S. Jacob C.G. Liu H. Sun X. Yang H. Nyunt M.M. Adams M. Zhou S. Xia Z. Ringwald P. Bustos M.D. Tang L. Plowe C.V. A single mutation in K13 predominates in Southern China and is associated with delayed clearance of Plasmodium falciparum following artemisinin treatment.J Infect Dis. 2015; 212: 1629-1635Crossref PubMed Scopus (111) Google Scholar, 12Ariey F. Witkowski B. Amaratunga C. Beghain J. Langlois A.C. Khim N. Kim S. Duru V. Bouchier C. Ma L. Lim P. Leang R. Duong S. Sreng S. Suon S. Chuor C.M. Bout D.M. Menard S. Rogers W.O. Genton B. Fandeur T. Miotto O. Ringwald P. Le Bras J. Berry A. Barale J.C. Fairhurst R.M. Benoit-Vical F. Mercereau-Puijalon O. Menard D. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.Nature. 2013; 505: 50-55Crossref PubMed Scopus (1285) Google Scholar, 13Tun K.M. Imwong M. Lwin K.M. Win A.A. Hlaing T.M. Hlaing T. Lin K. Kyaw M.P. Plewes K. Faiz M.A. Dhorda M. Cheah P.Y. Pukrittayakamee S. Ashley E.A. Anderson T.J. Nair S. McDew-White M. Flegg J.A. Grist E.P. Guerin P. Maude R.J. Smithuis F. Dondorp A.M. Day N.P. Nosten F. White N.J. Woodrow C.J. Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker.Lancet Infect Dis. 2015; 15: 415-421Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 14Menard D. Khim N. Beghain J. Adegnika A.A. Shafiul-Alam M. Amodu O. et al.A worldwide map of Plasmodium falciparum K13-propeller polymorphisms.N Engl J Med. 2016; 374: 2453-2464Crossref PubMed Scopus (354) Google Scholar Several reports also suggested that resistance emerged at least in part as independent events.15Takala-Harrison S. Jacob C.G. Arze C. Cummings M.P. Silva J.C. Dondorp A.M. et al.Independent emergence of Plasmodium falciparum artemisinin resistance mutations in Southeast Asia.J Infect Dis. 2014; 211: 670-679Crossref PubMed Scopus (303) Google Scholar, 16Miotto O. Amato R. Ashley E.A. MacInnis B. Almagro-Garcia J. Amaratunga C. et al.Genetic architecture of artemisinin-resistant Plasmodium falciparum.Nat Genet. 2015; 47: 226-234Crossref PubMed Scopus (372) Google Scholar However, recently, microsatellite analysis indicates that a mutant lineage carrying the Kelch13 PF3D7_1343700: p.Cys580Tyr single-nucleotide polymorphism (SNP) spread from Cambodia to Thailand, Laos, and Vietnam.17Imwong M. Suwannasin K. Kunasol C. Sutawong K. Mayxay M. Rekol H. Smithuis F.M. Hlaing T.M. Tun K.M. van der Pluijm R.W. Tripura R. Miotto O. Menard D. Dhorda M. Day N.P.J. White N.J. Dondorp A.M. The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: a molecular epidemiology observational study.Lancet Infect Dis. 2017; 17: 491-497Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 18Imwong M. Hien T.T. Thuy-Nhien N.T. Dondorp A.M. White N.J. Spread of a single multidrug resistant malaria parasite lineage (PfPailin) to Vietnam.Lancet Infect Dis. 2017; 17: 1022-1023Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar This is a continuously evolving situation, and it is unclear how and if this parasite lineage will continue to spread. For previous generations of antimalarials, resistance arose from the development and subsequent spread of one or more parasite SNPs. To monitor and prevent their spread, these SNPs can be detected with molecular assays, like sequencing or real-time PCR. However, the tests are relatively expensive, are labor intensive, and are generally performed in central laboratories. We sought to make SNP genotyping for antimalarial drug resistance more accessible outside of central laboratories by developing an assay that has the potential to be performed directly on blood in a point-of-care setting. To demonstrate proof of principle for the detection of drug resistance–associated SNPs, we tested our design with the well-characterized SNP associated with chloroquine resistance, c.227A>C (p.Lys76Thr) of the P. falciparum chloroquine resistance transporter (CRT) gene.19Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. Sidhu A.B. Naude B. Deitsch K.W. Su X.Z. Wootton J.C. Roepe P.D. Wellems T.E. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance.Mol Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar, 20Basco L.K. Ringwald P. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon.J Infect Dis. 2001; 183: 1828-1831Crossref PubMed Scopus (74) Google Scholar A common approach for PCR-based SNP identification uses hydrolysis probes because it can be performed in any laboratory equipped with a real-time PCR instrument. This technique relies on a large differential melting temperature between the probes specific for each polymorphism. The perfectly matched probe has a higher melting temperature for its target compared with the other mismatched probe.21Kim S. Misra A. SNP genotyping: technologies and biomedical applications.Annu Rev Biomed Eng. 2007; 9: 289-320Crossref PubMed Scopus (425) Google Scholar The difference in melting temperatures can be accomplished by using various probe modifications, like the minor groove binder (MGB).22Kutyavin I.V. Afonina I.A. Mills A. Gorn V.V. Lukhtanov E.A. Belousov E.S. Singer M.J. Walburger D.K. Lokhov S.G. Gall A.A. Dempcy R. Reed M.W. Meyer R.B. Hedgpeth J. 3'-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.Nucleic Acids Res. 2000; 28: 655-661Crossref PubMed Scopus (648) Google Scholar In the present study, locked nucleic acids (LNAs) were incorporated in the hydrolysis probes because they, like the MGB, raise the melting temperature of the probes, resulting in preferential binding of the wild-type probe to the wild-type target and the mutant probe to the mutant target.23Mouritzen P. Nielsen A.T. Pfundheller H.M. Choleva Y. Kongsbak L. Moller S. Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).Expert Rev Mol Diagn. 2003; 3: 27-38Crossref PubMed Scopus (90) Google Scholar In the case of LNAs, this is achieved by a methylene bridge within the ribose sugar of the nucleotide, resulting in a more rigid or locked conformation. This structural modification does not alter its participation in Watson-Crick base pairing, but does enhance the probe's affinity for its complementary target.24Braasch D.A. Corey D.R. Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.Chem Biol. 2001; 8: 1-7Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar The increase of the oligonucleotide melting temperature by LNAs provides a critical advantage for SNP genotyping and has been exploited for several assays, including Chlamydia pneumoniae genotyping.23Mouritzen P. Nielsen A.T. Pfundheller H.M. Choleva Y. Kongsbak L. Moller S. Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).Expert Rev Mol Diagn. 2003; 3: 27-38Crossref PubMed Scopus (90) Google Scholar, 25Rupp J. Solbach W. Gieffers J. Single-nucleotide-polymorphism-specific PCR for quantification and discrimination of Chlamydia pneumoniae genotypes by use of a "locked" nucleic acid.Appl Environ Microbiol. 2006; 72: 3785-3787Crossref PubMed Scopus (16) Google Scholar, 26Johnson M.P. Haupt L.M. Griffiths L.R. Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR.Nucleic Acids Res. 2004; 32: e55Crossref PubMed Scopus (130) Google Scholar, 27Simeonov A. Nikiforov T.T. Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection.Nucleic Acids Res. 2002; 30: e91Crossref PubMed Scopus (99) Google Scholar, 28Latorra D. Campbell K. Wolter A. Hurley J.M. Enhanced allele-specific PCR discrimination in SNP genotyping using 3' locked nucleic acid (LNA) primers.Hum Mutat. 2003; 22: 79-85Crossref PubMed Scopus (181) Google Scholar In addition to developing the molecular tools for SNP genotyping, our goal was to apply them within a simplified assay design appropriate for a low-resource setting. To avoid the complexities of sample preparation, a PCR-based assay that can be performed directly on whole blood was designed. Blood poses several obstacles for real-time PCR: it inhibits PCR amplification,29Al-Soud W.A. Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells.J Clin Microbiol. 2001; 39: 485-493Crossref PubMed Scopus (708) Google Scholar, 30Abu Al-Soud W. Radstrom P. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat.J Clin Microbiol. 2000; 38: 4463-4470Crossref PubMed Google Scholar and its absorption spectra can interfere with the fluorescent reporter dyes.31Bosschaart N. Edelman G.J. Aalders M.C. van Leeuwen T.G. Faber D.J. A literature review and novel theoretical approach on the optical properties of whole blood.Lasers Med Sci. 2014; 29: 453-479Crossref PubMed Scopus (274) Google Scholar Several manufacturers have developed Taq and other DNA polymerases to be highly resistant to PCR inhibitors, which are often referred to as robust polymerases.32Abu Al-Soud W. Radstrom P. Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples.Appl Environ Microbiol. 1998; 64: 3748-3753Crossref PubMed Google Scholar, 33Kermekchiev M.B. Kirilova L.I. Vail E.E. Barnes W.M. Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples.Nucleic Acids Res. 2009; 37: e40Crossref PubMed Scopus (187) Google Scholar, 34Trombley Hall A. McKay Zovanyi A. Christensen D.R. Koehler J.W. Devins Minogue T. Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.PLoS One. 2013; 8: e73845Crossref PubMed Scopus (47) Google Scholar For Taq polymerase, this can be accomplished by an N-terminal deletion that enhances performance, but at the cost of removing the 5′→3′ exonuclease activity required for cleavage of hydrolysis probes.33Kermekchiev M.B. Kirilova L.I. Vail E.E. Barnes W.M. Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples.Nucleic Acids Res. 2009; 37: e40Crossref PubMed Scopus (187) Google Scholar, 35Lawyer F.C. Stoffel S. Saiki R.K. Myambo K. Drummond R. Gelfand D.H. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.J Biol Chem. 1989; 264: 6427-6437PubMed Google Scholar In addition, although MGB probes with the reporter dyes 6-FAM (FAM) and VIC have been developed for c.227A>C (p.Lys76Thr) genotyping, they are not ideal for use with blood because of the high overlap between blood absorption and the reporter dyes' excitation and emission wavelengths.36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google Scholar The limitations associated with blood were overcome by incorporating two key changes to the PCR reagents: a robust Taq polymerase optimized for use with blood and the use of the fluorescent reporter dyes Cy5 and TEX615, which emit at sufficiently long wavelengths to reduce the optical interference from blood. The assay was also evaluated using Adaptive PCR, a previously described real-time PCR platform that uses left-handed DNA (L-DNA) additives to monitor the reaction for more reliable point-of-care performance.37Adams N.M. Gabella W.E. Hardcastle A.N. Haselton F.R. Adaptive PCR based on hybridization sensing of mirror-image l-DNA.Anal Chem. 2017; 89: 728-735Crossref PubMed Scopus (11) Google Scholar The P. falciparum reference line 3D7 was cultured, as previously described.38Sandlin R.D. Fong K.Y. Wicht K.J. Carrell H.M. Egan T.J. Wright D.W. Identification of beta-hematin inhibitors in a high-throughput screening effort reveals scaffolds with in vitro antimalarial activity.Int J Parasitol Drugs Drug Resist. 2014; 4: 316-325Crossref PubMed Scopus (30) Google Scholar Genomic DNA from the 3D7 parasite culture was extracted with the Qiagen (Germantown, MD) DNeasy Blood and Tissue kit, according to the manufacturer's protocol. The P. falciparum line 7G8 genomic DNA (MRA-926G, MR4; BEI Resources, Manassas, VA) was obtained through the Malaria Research and Reference Reagent Resource Center. Human whole blood with citrate phosphate dextrose anticoagulant was purchased from Bioreclamation IVT (Westbury, NY). For experiments using packed red blood cells (RBCs), the cells were washed in phosphate-buffered saline and used at a final concentration of 0.8%, which is equivalent to 2% whole blood for a 40% hematocrit. Ultramer DNA oligonucleotides containing the 166-base wild-type and the c.227A>C (p.Lys76Thr) mutant CRT (PlasmoDB Gene identification: PF3D7_0709000; https://plasmodb.org, last accessed February 1, 2019) amplicons were synthesized by Integrated DNA Technologies (Coralville, IA). All of the primer and probe sequences are shown in Table 1. The custom MGB probes (pfcrt_76K and pfcrt_76T) were supplied in a premixed solution with the primers pfcrt_F and pfcrt_R from Applied Biosystems (Foster City, CA). The MGB probes and their primers have been previously described.36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google Scholar The 5′ ends of the wild-type and mutant MGB probes were labeled with FAM and VIC fluorescent reporter dyes, respectively. The MGB quencher was located on their 3′ ends.Table 1Sequences of Oligonucleotides Used for Real-Time PCRNameDescriptionSequence∗Locked nucleic acid bases are preceded by a plus sign (+).pfcrt_F36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google ScholarForward primer5′-TGGTAAATGTGCTCATGTGTTT-3′pfcrt_R36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google ScholarReverse primer5′-AGTTTCGGATGTTACAAAACTATAGT-3′pfcrt_76K36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google ScholarWild-type probe with MGB5′-6-FAM-TGTGTAATGAATAAAATTTTTGCTAA-MGB-3′pfcrt_76T36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google ScholarMutant probe with MGB5′-VIC-TGTGTAATGAATACAATTTTTGCTAA-MGB-3′LNA_W (ML045)Wild-type probe with LNAs5′-Cy5-AT+G+AA+T+A+A+AAT+TTT+T+GC-IABRQSP-3′LNA_M (ML047)Mutant probe with LNAs5′-TEX615-GTAATGAA+T+A+C+AA+TT+TTTGCT-IABRQSP-3′ML040Wild-type probe with LNAs (candidate 1)5′-Cy5-TGTGTAATGAA+T+A+A+AA+TTTTTG+CT-IABRQSP-3′ML044Wild-type probe with LNAs (candidate 2)5′-Cy5-AT+G+AA+T+A+A+AA+TT+TTT+GCT-IABRQSP-3′ML046Wild-type probe with LNAs (candidate 4)5′-Cy5-T+G+AA+T+A+A+AAT+TTT+T+GCT-IABRQSP-3′ML036Mutant probe with LNAs (candidate 1)5′-HEX-TGTGTAATGAA+T+A+C+AA+TTTTTG+CTA-IABKFQ-3′ML037Mutant probe with LNAs (candidate 2)5′-HEX-TGTGTAATGAA+T+A+C+AA+T+TTTTGCTA-IABKFQ-3′IABKFQ, Iowa Black FQ; IABRQSP, Iowa Black RQ; LNA, locked nucleic acid; MGB, minor groove binder.∗ Locked nucleic acid bases are preceded by a plus sign (+). Open table in a new tab IABKFQ, Iowa Black FQ; IABRQSP, Iowa Black RQ; LNA, locked nucleic acid; MGB, minor groove binder. A second set of pfcrt_F and pfcrt_R primers as well as the LNA probes were synthesized by Integrated DNA Technologies. Wild-type probes incorporating between six and nine LNA bases were labeled with Cy5 on the 5′ end. The probes for the mutant alleles were labeled with either HEX or TEX615. The FAM and HEX dyes were quenched with a 3′ Iowa Black FQ quencher and the TEX615 and Cy5 dyes with a 3′ Iowa Black RQ quencher. Each was tested for its SNP discrimination potential in a competition assay with each probe from the other allele. The probes with the highest ratio of perfect-match-to-mismatch end point fluorescence, labeled LNA_W and LNA_M in Table 1, were chosen for further study. They contained nine and six locked nucleotides, respectively, and were synthesized with a 5′ reporter dye (Cy5 or TEX615) and a 3′ Iowa Black RQ quencher. L-DNA enantiomer oligonucleotides were synthesized by Biomers (Ulm, Germany). To monitor primer annealing, L-DNA enantiomers of the pfcrt_F primer and its reverse complement were synthesized. The previously described 77-mer sequence derived from Mycobacterium tuberculosis (labeled as TB77) was synthesized and used to monitor melting of the amplicon.37Adams N.M. Gabella W.E. Hardcastle A.N. Haselton F.R. Adaptive PCR based on hybridization sensing of mirror-image l-DNA.Anal Chem. 2017; 89: 728-735Crossref PubMed Scopus (11) Google Scholar For reactions with the LNA probes, each 25-μL reaction contained 1× Taq Mutant Reaction Buffer (DNA Polymerase Technology, St. Louis, MO), 200 nmol/L of each dNTP (Sigma-Aldrich, St. Louis, MO), 400 nmol/L of each probe, 900 nmol/L of each primer, 0.25 μL of OmniTaq 3 DNA polymerase (DNA Polymerase Technology), and up to 3% whole blood or washed RBCs from the P. falciparum (strain 3D7) parasite culture. The reactions with MGB probes were composed identically with the exception of the probes and primers, which were supplied in a 20× mixture by the manufacturer for a final concentration of 200 and 900 nmol/L, respectively. In addition, the target DNA was added at 107 copies for the synthesized oligonucleotides and 8 ng (approximately 3 × 105 copies) for the genomic DNA. Two PCR instruments were used for this study: the Rotor-Gene Q (Qiagen), a traditional PCR instrument; and the recently described Adaptive PCR instrument. The latter uses fluorescently labeled L-DNA analogs of the primers and amplicon to identify heating and cooling switch points by optically monitoring the annealing of L-DNA primers and melting of the L-DNA amplicon, respectively.37Adams N.M. Gabella W.E. Hardcastle A.N. Haselton F.R. Adaptive PCR based on hybridization sensing of mirror-image l-DNA.Anal Chem. 2017; 89: 728-735Crossref PubMed Scopus (11) Google Scholar For the Rotor-Gene instrument, each reaction was performed with the following conditions: an initial 95°C hold for 3 minutes, followed by 45 cycles at 95°C for 15 seconds and 62°C for 15 seconds. On the Adaptive PCR instrument, the 3-minute initial denaturation was performed by programming LabVIEW software version 2017 (National Instruments, Austin, TX) to monitor the fluorescence signal of labeled L-DNA analogs of the TB77 amplicon while heating and then maintaining the fluorescence signal once the melt plateau was reached. The program maintained the melted state for 3 minutes by turning the heater on if the fluorescence signal fell below the melt state and turning the heater off once the fluorescence reached the melt state. Validation studies with a thermocouple inserted into the reaction tube confirmed that the hold temperature maintained approximately 88°C ± 2°C for the denaturation step. The samples were then cooled until the L-DNA analog of the pfcrt_F primer annealed to its target. The instrument then began heating until the L-DNA TB77 amplicon strands separated. This was repeated for a total of 40 cycles. The end point fluorescence values for the allelic discrimination plots were taken from PCR cycle number 40, regardless of the instrument. Our goal was to streamline SNP genotyping assays to make them more compatible for malaria fieldwork. One of the main bottlenecks is DNA extraction because it is time and labor intensive, and consequently, is usually performed in a traditional laboratory setting. Aside from the inhibitors found in blood, the excitation and emission spectra of commonly used probe reporter dyes are also incompatible with the absorbance spectra of blood. To overcome this problem, the Cy5 and TEX615 reporter dyes were assessed because they excite and emit in the orange-red end of the spectrum, where blood absorbance is lower. The strategy for evaluating the probes in blood was to increase the experimental complexity one step at a time. First, the LNA probes were compared with previously described MGB genotyping probes36Wilson P.E. Kazadi W. Kamwendo D.D. Mwapasa V. Purfield A. Meshnick S.R. Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.Acta Trop. 2005; 93: 97-106Crossref PubMed Scopus (32) Google Scholar by using amplicon-length oligonucleoti
Referência(s)