title: Development of a thermostabilised triplex LAMP assay with dry-reagent four target lateral flow dipstick for detection of Entamoeba histolytica and non-pathogenic Entamoeba spp.
abstract: This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: pcfoo12@gmail.com.
authors.forename: Phiaw Chong
authors.initials: PC
authors.lastname: Foo
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: yeancyn@yahoo.com.
authors.forename: Yean Yean
authors.initials: YY
authors.lastname: Chan
authors.affiliation: Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, City Campus, Pengkalan Chepa, Locked Bag 36, 16100 Kota Bharu, Kelantan, Malaysia. Electronic address: maizan.m@umk.edu.my.
authors.forename: Maizan
authors.initials: M
authors.lastname: Mohamed
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: kinnywong84@gmail.com.
authors.forename: Weng Kin
authors.initials: WK
authors.lastname: Wong
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: nurulnajian@yahoo.com.
authors.forename: A B
authors.initials: AB
authors.lastname: Nurul Najian
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia; Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: limbh493@gmail.com.
authors.forename: Boon Huat
authors.initials: BH
authors.lastname: Lim
mesh_terms: D015374:Biosensing Techniques; D016054:DNA, Protozoan / Q000032:analysis; D004747:Entamoeba / Q000302:isolation & purification*; D004748:Entamoeba histolytica / Q000302:isolation & purification*; D004749:Entamoebiasis / Q000175:diagnosis; D005243:Feces / Q000469:parasitology; D006046:Gold; D006801:Humans; D007118:Immunoassay; D053768:Metal Nanoparticles; D021141:Nucleic Acid Amplification Techniques; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D006046:Gold
keywords: DNA lateral flow biosensor; Entamoeba histolytica; Isothermal amplification; Multiplex LAMP; Thermostabilization