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In brief:  LAMP assays can be run under isothermal conditions at 65°C.  Due to the short test runtime compared to classical PCR and the possibility of evaluation in a 384-well format on a plate reader, a significant increase of the global test capacity of SARS-CoV-2 could be achieved.

The spread of the SARS-CoV-2 pandemic could be slowed down by an increase in global testing approaches. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) describes an alternative method to amplify and detect viral RNA. LAMP recognizes the target RNA by several sequences and amplifies it with comparable sensitivity and specificity to PCR-based methods.1,2 The Bst polymerase works optimally at 65°C, meaning that the LAMP assay can be performed under isothermal conditions in only 30 min.1  Since the need for temperature cycles is bypassed, the assay can be run on a plate reader with heating function up to 65°C.

Assay principle of the colorimetric LAMP assay

A thermophilic polymerase and primer sets recognize the SARS-CoV-2 nucleic acid sequence at different positions. Nucleic acid amplification leads to the release of hydrogen ions, shifting the pH toward the acidic side. Consequently, the absorbance maximum of an included pH-sensitive dye shifts also (here from 415 nm to 560 nm). This shift in absorbance is quantified on a FLUOstar® Omega plate reader (Figure 1).

Figure 1. Assay principle

Simultaneous incubation and analysis of the LAMP assay

The colorimetric SARS-CoV-2 LAMP assay (NEB, #E2019S) was performed on a FLUOstar Omega to illustrate the reader’s capability to simultaneously incubate and acquire data kinetically. Accordingly, the assay was incubated at 65°C for 60 min in a 384-well plate with a final reaction volume of 5 µL/well using the positive control (n-gene) diluted 1:10. Absorbance at 415 nm and that at 560 nm were simultaneously detected in the whole plate.

Plate reader-based detection of LAMP assays paves the way for high-throughput screening

The ∆OD (415 nm-560 nm) of the positive controls started to increase substantially at about 18 min after the assay’s start. The negative controls also displayed an increase of ∆OD which is the result of spurious amplification products—a well-known event in LAMP assays. The kinetic monitoring of the amplification on the FLUOstar Omega plate reader allows to define the ideal measurement window and thereby optimal cut off points to discriminate positive from negative samples (orange frame, Figure 2A). As the UV/Vis spectrometer of the FLUOstar Omega simultaneously detects at 415 nm and 560 nm, frequent measurement time points can be obtained (133s/384-well plate), strengthening the confidence in the obtained results.  While positive controls reach maximum slope after about 19 min, negative samples need about 39 min (Figure 2B, evaluation was performed using preset calculations of BMG LABTECH’s accompanying MARS software).

Figure 2. (A) Kinetic measurement of the SARS-CoV-2 LAMP assay over 60 min in ∆OD.2 The optional measurement window is marked in orange (B) Time to maximum slope of positive and negative controls, derived from kinetic measurement over 60 min, n=4, ***p<.001.

Running the LAMP assay on a microplate reader enables users to test samples in under 40 minutes. The LAMP assay as a rapid diagnostic method therefore offers an opportunity to work in high throughput, increasing diagnostic capacity as an alternative or in ßaddition to PCR techniques.

 

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