TITLE: INSIGHT: a scalable isothermal NASBA-based platform for COVID-19 diagnosis
AUTHORS: Qianxin  Wu, Chenqu Suo, Tom  Brown, Tengyao  Wang, Sarah Teichmann, Andrew Bassett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We present here INSIGHT (Isothermal NASBA-Sequencing based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a combination of an isothermal NASBA reaction and next generation sequencing. From commercially acquired human saliva with spiked-in viral RNA as input, the first stage employs isothermal amplification of viral RNA to give a rapid result in one to two hours, using either fluorescence detection or a dipstick readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. In the first stage, fluorescent viral RNA detection can be consistently achieved at 10-100 copies per 20 μl reaction. The second stage pools post-amplification barcoded products from multiple samples for scalable sequencing that could be centralised, to further improve the accuracy of the test in a massively parallel way. Our two-stage testing strategy is suitable for further development into a home-based or point-of-care assay, and is potentially scalable to population level.</div></div>

[STEPS]
?. Lysis of saliva samples
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							Mix crude saliva at 1:1 ratio with QuickExtract DNA Extraction Solution. Incubate at 95 °C for 5 min to ensure complete lysis of virus and inactivation of proteinase K. 
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?. NASBA reaction
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							Take 1 μl from the product of Step 1 and add into the NASBA reaction mixture to make a total volume of 20 μl. Reaction mixture can either be prepared in-house or from the Life Sciences NASBA liquid kit (see tables below).
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							A fluorescence plate reader can be used to monitor the reaction in real-time, or as an endpoint assay.
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							  ABCD1 vol.stock conc.conc. in RM2sample1 μl  3primers*1 μl0.5 μM each25 nM each4water +/- beacon3 μl 20 nM for beacon5buffer (NECB-24)6.7 μl  6nucleotide (NECN-24)3.3 μl  7enzyme mix (NEC-1-24)5 μl  8total volume20 μl    ABCD1 vol.stock conc.conc. in RM2sample1 μl  3primers*1 μl0.5 μM each25 nM each4water +/- beacon4 μl 20 nM for beacon5buffer with DMSO*5 μl  6nucleotide mix*4 μl  7enzyme mix*5 μl  8total volume20 μl    ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4*120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6DMSO450 μl100%11%7water247.9 μl  8total volume1000 μl    ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6water697.9 μl  7total volume1000 μl    ABCD1  vol.stock conc.conc. in RM2dNTP0.22 μl each100 mM1 mM each3NTP0.88 μl each100 mM4 mM each4total volume4.4 μl    ABCD1  vol.stock conc.conc. in RM2diluted Rnase H0.17 μl500 U/ml3.75 U/ml3Photoscript RT 0.28 μl200000 U/ml2500 U/ml4T7 polymerase 2.75 μl50000 U/ml6250 U/ml5BSA0.13 μl20 mg/ml0.12 mg/ml6buffer without DMSO1.78 μl  7water0.40 μl  8total volume5.5 μl    ABC1  vol.stock conc.2Rnase H5 μl5000 U/ml3BSA (0.48mg/ml)1.2 μl20 mg/ml4buffer without DMSO16.67 μl 5water27.13 μl 6total volume50 μl
ABCD1 vol.stock conc.conc. in RM2sample1 μl  3primers*1 μl0.5 μM each25 nM each4water +/- beacon3 μl 20 nM for beacon5buffer (NECB-24)6.7 μl  6nucleotide (NECN-24)3.3 μl  7enzyme mix (NEC-1-24)5 μl  8total volume20 μl
ABCD1 vol.stock conc.conc. in RM2sample1 μl  3primers*1 μl0.5 μM each25 nM each4water +/- beacon4 μl 20 nM for beacon5buffer with DMSO*5 μl  6nucleotide mix*4 μl  7enzyme mix*5 μl  8total volume20 μl
ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4*120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6DMSO450 μl100%11%7water247.9 μl  8total volume1000 μl
ABCD1  vol.stock conc.conc. in RM2Tris-HCl pH 8.4120 μl1 M40 mM3MgCl239.6 μl1 M13.2 mM4KCl112.5 μl2 M75 mM5DTT 30 μl1 M10 mM6water697.9 μl  7total volume1000 μl
ABCD1  vol.stock conc.conc. in RM2dNTP0.22 μl each100 mM1 mM each3NTP0.88 μl each100 mM4 mM each4total volume4.4 μl
ABCD1  vol.stock conc.conc. in RM2diluted Rnase H0.17 μl500 U/ml3.75 U/ml3Photoscript RT 0.28 μl200000 U/ml2500 U/ml4T7 polymerase 2.75 μl50000 U/ml6250 U/ml5BSA0.13 μl20 mg/ml0.12 mg/ml6buffer without DMSO1.78 μl  7water0.40 μl  8total volume5.5 μl
ABC1  vol.stock conc.2Rnase H5 μl5000 U/ml3BSA (0.48mg/ml)1.2 μl20 mg/ml4buffer without DMSO16.67 μl 5water27.13 μl 6total volume50 μl
?. Detection with lateral flow dipstick (if desired)For detection with lateral flow assay, Bio-11-UTP can be added into the NASBA nucleotide mixture in Step 2 at a final concentration of 0.5 μM. At the end of the NASBA reaction, RNA is purified from the end-product using Direct-zol RNA Miniprep kit, and eluted with 30 μl of RNase free water. After purification, 4.2 μl of purified RNA is mixed with 1.8 μl of FAM labelled RNA capture oligo and 84 μl of PCRD extraction buffer (see table below). Take 75 μl of mix to the sample well of a PCRD test cassette. Results will be shown within 10 min.
?. Sequencing stage
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							To allow for pooled sequencing of NASBA reaction end products, barcode sequences are added upstream of each of the forward and reverse primers (Figure 3a). In addition, an Illumina sequencing adaptor is added upstream of the forward primer barcode sequence as a universal PCR handle (see Materials and Reagents for the exact sequence). 
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							Uniquely barcoded NASBA end products from different samples are pooled and purified. A one step PCR can be carried out at local sequencing centres using universal P5 and P7 primers (Figure 3b). Here, if needed, another layer of indexing barcodes can be added to further increase the multiplexing capacity.
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?. Detection with lateral flow dipstick (if desired)
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							For detection with lateral flow assay, Bio-11-UTP can be added into the NASBA nucleotide mixture in Step 2 at a final concentration of 0.5 mM. At the end of the NASBA reaction, RNA is purified from the end-product using Direct-zol RNA Miniprep kit, and eluted with 30 μl of RNase free water. After purification, 4.2 μl of purified RNA is mixed with 1.8 μl of FAM labelled RNA capture oligo and 84 μl of PCRD extraction buffer. Take 75 μl of mix to the sample well of a PCRD test cassette. Results will be shown within 10 min. 
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?. Sequencing stage
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							To allow for pooled sequencing of NASBA reaction end products, barcode sequences are added upstream of each of the forward and reverse primers. In addition, an Illumina sequencing adaptor is added upstream of the forward primer barcode sequence as a universal PCR handle (see Materials and Reagents for the exact sequence). 
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							Uniquely barcoded NASBA end products from different samples are pooled and purified. A one step PCR can be carried out at local sequencing centres using universal P5 and P7 primers. Here, if needed, another layer of indexing barcodes can be added to further increase the multiplexing capacity.
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