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dc.contributor.authorSeevaratnam, Dushanth
dc.contributor.authorAnsah, Felix
dc.contributor.authorAniweh, Yaw
dc.contributor.authorAwandare, Gordon A
dc.contributor.authorHall, Lisa
dc.date.accessioned2022-07-05T01:01:38Z
dc.date.available2022-07-05T01:01:38Z
dc.date.issued2022-06-03
dc.identifier.issn1618-2642
dc.identifier.otherPMC9163865
dc.identifier.other35657389
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/338762
dc.description.abstractBacillus stearothermophilus large fragment (BSTLF) DNA polymerase is reported, isolated on silica via a fused R5 silica-affinity peptide and used in nucleic acid diagnostics. mCherry (mCh), included in the fusion construct, was shown as an efficient fluorescent label to follow the workflow from gene to diagnostic. The R5 immobilisation on silica from cell lysate was consistent with cooperative R5-specific binding of R52-mCh-FL-BSTLF or R52-mCh-H10-BSTLF fusion proteins followed by non-specific protein binding (including E. coli native proteins). Higher R5-binding could be achieved in the presence of phosphate, but phosphate residue reduced loop-mediated isothermal amplification (LAMP) performance, possibly blocking sites on the BSTLF for binding of β- and γ-phosphates of the dNTPs. Quantitative assessment showed that cations (Mg2+ and Mn2+) that complex the PPi product optimised enzyme activity. In malaria testing, the limit of detection depended on Plasmodium species and primer set. For example, 1000 copies of P. knowlesi 18S rRNA could be detected with the P.KNO-LAU primer set with Si-R52-mCh-FL-BSTLF , but 10 copies of P. ovale 18S rRNA could be detected with the P.OVA-HAN primer set using the same enzyme. The Si-immobilised BSTLF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R52-mCh-FL-BSTLF production was transferred from Cambridge to Accra and set up de novo for a trial with clinical samples. Different detection limits were found, targeting the mitochondrial DNA or the 18S rRNA gene for P. falciparum. The results are discussed in comparison with qPCR and sampling protocol and show that the Si-BSTLF polymerase can be optimised to meet the WHO recommended guidelines.
dc.description.sponsorshipRoyal Society International Collaboration Award, IC160089
dc.languageeng
dc.publisherSpringer Science and Business Media LLC
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourcenlmid: 101134327
dc.sourceessn: 1618-2650
dc.subjectMalaria
dc.subjectLAMP
dc.subjectLow Cost
dc.subjectBst Polymerase
dc.subjectLocal Production
dc.titleAnalysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis.
dc.typeArticle
dc.date.updated2022-07-05T01:01:35Z
prism.publicationNameAnal Bioanal Chem
dc.identifier.doi10.17863/CAM.86172
dcterms.dateAccepted2022-05-12
rioxxterms.versionofrecord10.1007/s00216-022-04131-2
rioxxterms.versionVoR
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidSeevaratnam, Dushanth [0000-0001-7302-8379]
dc.contributor.orcidAnsah, Felix [0000-0002-6928-2363]
dc.contributor.orcidAniweh, Yaw [0000-0002-8415-2727]
dc.contributor.orcidAwandare, Gordon A [0000-0002-8793-3641]
dc.contributor.orcidHall, Lisa [0000-0001-9572-9854]
dc.identifier.eissn1618-2650
pubs.funder-project-idRoyal Society (IC160089)
cam.issuedOnline2022-06-03


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International