| CPC B01L 7/52 (2013.01) [B01L 3/502715 (2013.01); C12Q 1/6832 (2013.01); C12Q 1/686 (2013.01); C12Q 1/701 (2013.01); G01N 33/54386 (2013.01); B01L 2300/126 (2013.01); B01L 2300/1805 (2013.01); B01L 2300/1844 (2013.01); G01N 2333/165 (2013.01)] | 16 Claims |
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1. A method for rapid and piecewise on-site analyte sample-based detection of pathogenic infection via a nucleic acid-based test following sample-to-result integration for any number of different stand-alone nucleic acid-based tests in a portable point of care (POC) based device comprising: providing a modular and scalable thermal control cum reaction unit including a heating block for controlled piecewise isothermal heating and cooling cycles including: at least one microcontroller based isothermal heating unit to accommodate any required number of stand-alone nucleic acid-based tests of the analyte samples following a RT LAMP reaction; and utilizing a cooperatively disposed POC colorimetric detector to perform steps comprising of:
(I) carrying out each of the nucleic acid-based tests for the analyte samples inside a respective movable enclosed reaction microchamber in the microcontroller based isothermal heating unit following the RT LAMP reaction with a RT LAMP reaction mixture, the RT LAMP reaction mixture comprising RT LAMP reaction specific primers, said analyte samples containing pathogenic RNA along with:
(a) a biotinylated forward inner primer (FIP-5′Bt); and
(b) a double modified DNA oligonucleotide probe harboring 6-fluorescein amidite and a di-deoxy nucleotide, providing for seamlessly integrating the steps of performing:
(i) an initial RT LAMP reaction for reverse transcription of the pathogenic RNA into cDNA in the presence of the biotinylated forward inner primer (FIP-5′Bt) to thereby generate amplified 5′biotinylated labelled target DNA; and
(ii) subsequently, conducting hybridization of the thus amplified 5′biotinylated labelled target DNA with the double modified DNA oligonucleotide probe harboring 6-fluorescein amidite and a di-deoxy nucleotide complementary to the loop regions of the 5′biotinylated labelled target DNA products within said enclosed reaction microchambers; and thereby generating:
(a) dual labelled RT LAMP-based hybridized reaction products with biotin and 6-fluorescein amidite attached and
(b) single labelled free probes,
wherein the RT LAMP-based hybridized reaction products have improved specificity and are free of contamination, free of concomitant amplification of the probe(s), free of non-specific signals or primer dimer formation
(II) dispensing the RT LAMP-based hybridized reaction products and the single labelled free probes to the cooperatively disposed POC colorimetric detector, the POC colorimetric detector comprising: a replaceable functionalized microfluidic paper substrate/strip; and a selectively functionalized surface plasmon resonating nanomaterial conjugated anti-fluorescein amidite antibody and immobilized bio-conjugate section for a desired colorimetric based detection as a lateral flow assay wherein the surface plasmon resonating nanomaterial conjugated anti-fluorescein amidite antibody binds specifically with the dual labelled RT LAMP-based hybridized reaction products thereby generating colorimetric changes in the immobilized bio-conjugate section for imaging indicative of detection of the pathogenic infection.
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