Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for...

Bacillus stearothermophilus large fragment (BST_LF) 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-BST_LF or R5₂-mCh-H10-BST_LF 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 BST_LF 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-BST_LF , 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 BST_LF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R5₂-mCh-FL-BST_LF 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-BST_LF polymerase can be optimised to meet the WHO recommended guidelines.