A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level

Abstract

Bacterial contamination accounts for more than half of food poisoning cases. Conventional methods such as colonycounting and general polymerase chain reaction are timeconsuming, instrument-dependent, and sometimes not accurate. Herein, we developed a novel one-pot toolbox with precision and ultra sensitivity (OCTOPUS) platform for foodborne pathogen detection based on the mechanism in which Cas12a nontarget binding unleashes its collateral DNase activity. We demonstrated its application on two widespread foodborne bacteria, namely, E. coli O157:H7 and Streptococcus aureus, using specific crRNA targeting rfbE and nuc gene, respectively. For better sensitivity, recombinase polymerase amplification (RPA) was integrated without product purification. This one-pot detection, that is, RPA reagent, crRNA, and ssDNA-FQ reporter are all in one tube with the subsequent addition of Cas12a enzyme, was able to detect genomic DNA at the attomolar level. It omits an extra cap-opening process to avoid practical inconvenience and possible crosssample contamination. Moreover, we demonstrated this platform for a real food matrix. A simple water boiling method for genome extraction together with one-pot assay achieved a limit of detection value of 1 CFU/mL in less than 50 min. This OCTOPUS technique integrates bacterial genome extraction, preamplification based on RPA, and Cas12a/crRNA cleavage assay.

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

“…To develop one-pot iSCAN detection system with AapCas12b, we employed different strategies; 5 μL of 5 μM AapCas12b was spotted to the walls of the tube ( Wang et al, 2020 ; Wang et al, 2019 ) or directly added to the initial 50 μL reaction mix, independently The initial 50 μL of reaction mix contains the complete RT-LAMP reagents such as 5 μL 10X isothermal amplification buffer, 5 μL 10X Primer Mix, 4 μL of 100 mM MgSO 4, 7 μL of 10 mM dNTP mix, 2 μL of Bst LF DNA polymerase (50 ng), and 2 μL of RTx reverse transciptase 4 ng/μl, together with 2.5 μL of 500 ng/μL salmon sperm DNA, 2 μL of viral (control or clinical sample) RNA, 5 μL of 5 μM specific (or non-specific) AacCas21b sgRNAs and 5 μL of 5 μM lateral flow cleavage reporter (​5′-​/56-FAM/TTATTATT/3Bio/​-3′​, IDT) and 5.5 μL nuclease-free water. After the addition of all reagents we incubated the reaction tubes horizontally in a water bath at 62 °C for 30 min and then spin down the tubes to mix the AapCas12b with the LAMP reaction and incubated the reaction tubes again at 62 °C for 30 min for further AapCas12b based collateral activity.…”

“…To develop one-pot iSCAN detection system with AapCas12b, we employed different strategies; 5 μ L of 5μM AapCas12b was spotted to the walls of the tube [44,45] RT-PCR was conducted by using the oligonucleotide primer/probe (Integrated DNA Technologies, catalog #10006606) and Superscript III one-step RT-PCR system with Platinum Taq Polymerase (catalog #12574-026) as per the manufacturer's protocol on extracted RNA sample. The RNA sample for SARS nCoV 2019 considered as positive when the cycle threshold (Ct) value for both E and N genes was ≤ 36 and negative when the Ct value was more than > 36 for both genes.…”

A general onepot-method for nucleic acid detection with CRISPR-Cas12a

“…With these innovations, we integrate the ampli cation and detection into a single reaction system, which is highly valuable as avoidance of uncapping is important for prevention of aerosol generation that easily causes false positivity. Previous studies adopted a physical separation method to combine nucleic acid ampli cation and Cas12a detection into a single tube 16,25 . Wang et.…”

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

“…To develop one-pot iSCAN detection system with AapCas12b, we employed different strategies; L of 5 μL of 5μM AapCas12b was spotted to the walls of the tube [44, 45] or directly added to the initial 50 μL reaction mix, independently. The initial 50 μL of reaction mix contains the complete RT-LAMP reagents such as 5 μL 10X isothermal amplification buffer, 5 μL 10X Primer Mix, 4 μL of 100 mM MgSO 4 , 7 μL of 10 mM dNTP mix, 2 μL of Bst LF DNA polymerase (50 ng), and 2 ul of RTx reverse transciptase 4 ng/μl, together with 2.5 μL of 500 ng/μL salmon sperm DNA, 2 μL of viral (control or clinical sample) RNA, 5 μL of 5 μM specific (or non-specific) AacCas21b sgRNAs and 5 μL of 5 μM lateral flow cleavage reporter (5'-/56-FAM/TTATTATT/3Bio/-3', IDT) and 5.5 μL nuclease-free water.…”