Rapid detection of measles virus RNA from clinical specimens by using RT-LAMP coupled with CRISPR/Cas12b via fluorescence and lateral flow biosensor readouts: A proof-of-concept study

ABSTRACT

Rapid laboratory confirmation of suspected cases is essential for measles control, but current methods require a complex laboratory infrastructure. We developed loop-mediated isothermal amplification coupled with CRISPR-Cas-mediated diagnostic (LAmCaD), a novel two-pot diagnostic platform that integrates rapid nucleic acid extraction, RT-LAMP amplification, and in-house purified AapCas12b as a rapid test to detect measles RNA that could be used in settings lacking laboratory infrastructure.LAmCaD is based on dual detection modalities, fluorescence and lateral flow biosensor readouts. The LAmCaD assay was evaluated for analytical sensitivity and specificity using RNA from Vero/hSLAM-grown measles virus, and diagnostic evaluation was performed using patient samples, compared with standard RT-PCR. The cross-genotype detection capability was assessed across epidemiologically relevant measles genotypes D8, D4, and B3.The in-house purified protein AapCas12b from Alicyclobacillus acidiphilus exhibited strong cis and trans cleavage activities, eliminating dependence on commercial enzyme preparations. The platform enables the use of clinical samples from patients through rapid nucleic acid extraction that eliminates the need for RNA purification steps, allowing direct use of extracted material in RT-LAMP reactions. RT-LAMP alone achieved analytical sensitivity of ∼103 copies, while the complete protocol detected measles virus at ∼105 copies by fluorescence and ∼104 copies by lateral flow detection. Diagnostic evaluation demonstrated sensitivity of 64.00 %, specificity of 92.59 %, and negative predictive value of 99.95 % with an overall accuracy of 92.56 %. ROC curve analysis revealed an AUC of 0.717, indicating fair discriminatory performance. The assay demonstrated moderate agreement with RT-PCR (κ = 0.6) and successfully identified genotypes D8, D4, and B3. The entire testing process took 90 min to complete.This proof-of-concept LAmCaD platform establishes the foundation for a cost-effective, field-deployable diagnostic test without relying on commercial enzymes or complex sample processing. The platform could facilitate the rapid confirmation of measles cases in resource-limited and/or remote settings, thereby contributing to global measles elimination goals. Conducted within the WHO South-East Asia Region, this study is particularly relevant to the region's 2023 elimination target, addressing current surveillance gaps and specimen transport challenges that hinder efforts to eliminate the disease.