CoLAMP: CRISPR-based one-pot loop-mediated isothermal amplification enables at-home diagnosis of SARS-CoV-2 RNA with nearly eliminated contamination utilizing amplicons depletion strategy.

Rapid point-of-care diagnostics, essential in settings such as airport on-site testing and home-based screening, displayed important implications for infectious disease control during the SARS-CoV-2 outbreak. However, the deployment of simple and sensitive assays in real-life scenarios still faces the concern of aerosol contamination. Here, we report an amplicon-depleting CRISPR-based one-pot loop-mediated isothermal amplification (CoLAMP) assay for point-of-care diagnosis of SARS-CoV-2 RNA. In this work, AapCas12b sgRNA is designed to recognize the activator sequence sited in the loop region of the LAMP product, which is crucial for exponential amplification. By destroying the aerosol-prone amplifiable products at the end of each amplification reaction, our design can significantly reduce the amplicons contamination that causes false positive results in point-of-care diagnostics. For at-home self-testing, we designed a low-cost sample-to-result device for fluorescence-based visual interpretation. As well, a commercial portable electrochemical platform was deployed as a proof-of-concept of ready-to-use point-of-care diagnostic systems. The field deployable CoLAMP assay can detect as low as 0.5 copies/µL of SARS-CoV-2 RNA in clinical nasopharyngeal swab samples within 40 min without the need for specialists for its operation.

Disposable and low-cost pen-like sensor incorporating nucleic-acid amplification based lateral-flow assay for at-home tests of communicable pathogens.

Rapid at-home test is a good alternative to the gold standard quantitative polymerase chain reaction (qPCR) for early identification and management of infected individuals in pandemic. However, the currently available at-home rapid antigen kits and nucleic acid tests (NATs) are prone to false results. Although some CRISPR-mediated NATs enhanced accuracy, long turnaround time (ca. 1 h) and aerosol contamination due to additional open-lid reaction hinder its applicability for self-tests. Moreover, the accuracy of at-home NATs is also impacted by interference of sample matrix due to lack of sample purification. Here we report a Fast, Low-cost, Aerosol contamination-free and Sensitive molecular assay for at-Home tests of communicable pathogens (FLASH) incorporating oLAMP, a recently reported isothermal and target-specific NATs by our group, and a visible lateral-flow readout. The integrated platform enabled sample-to-result SARS-CoV-2 RNA detection in 20-30 min achieving a sensitivity of 0.5 copies/µL in a blinded experiment with a high accuracy comparable with the qPCR. Its prototype consists of two disposable pen-like instruments for single-step sample preparation and contamination-free NATs, respectively. The simplified workflow of the FLASH enabled detection to be readily conducted by untrained users for at-home tests. All in all, the FLASH prototype demonstrates itself to be a promising home-use assay platform for effective mitigation of the pandemic.

Rational design of allosterically regulated toehold mediated strand displacement circuits for sensitive and on-site detection of small molecule metabolites.

Development of small molecule biosensors enables rapid and de-centralized small molecule detection that meets the demands of routine health monitoring and rapid diagnosis. Among them, allosteric transcription factor (aTF)-based biosensors have shown potential in modular design of small molecule detection platforms due to their ligand-regulated DNA binding activity. Here, we expand the capabilities of a biosensor that leverages the aTF-based regulation of toehold-mediated strand displacement (TMSD) circuits for uric acid (UA) detection in non-invasive salivary samples by utilizing the UA-responsive aTF HucR. The impact of the low ligand affinity of the native HucR was addressed by engineering a two-pass TMSD circuit with in silico rational design. This combined strategy achieved enrichment of the output signal and overcame the negative impact of the matrix effect on the sensitivity and overall response of the biosensor when using real samples, which enabled semi-quantitative detection in the normal salivary UA levels. As well, enhancements provided by the two-pass design halved the turnaround time to less than 15 minutes. To sum up, the two-cycle DNA circuit design enabled aTF-based simple, rapid and one-step non-invasive salivary UA detection, showing its potential in metabolite detection for health monitoring.

Improved defined approaches for predicting skin sensitization hazard and potency in humans.

Since the EU banned animal testing for cosmetic products and ingredients in 2013, many defined approaches (DA) for skin sensitization assessment have been developed. Machine learning models were shown to be effective in DAs, but the predictivity might be affected by data imbalance (i.e. more numbers of sensitizers than non-sensitizers) and limited information in the databases. To improve the predictivity of DAs, here we attempted to apply data-rebalancing ensemble learning (bagging with support vector machine (SVM)) and a novel and comprehensive Cosmetics Europe database. For predicting human hazard and three-class potency, 12 models were built for each using a training set of 96 substances and a test set of 32 substances from the database. The model with the highest accuracy for predicting hazard (90.63% for the test set and 88.54% for the training set, named hazard-DA) used the SVM-bagging with combinations of all variables (V6), while the model with the highest accuracy for predicting potency (68.75% for the test set and 82.29% for the training set, named potency-DA) used SVM alone. Both DAs showed higher performance than LLNA and other machine-learning-based DAs, and the potency-DA could provide more in-depth assessment. Those findings indicated that SVM-bagging-based DAs provided enhanced predictivity for hazard assessment by further data rebalancing. Meanwhile, the effect of imbalanced data might be offset by more detailed categorization of sensitizers for potency assessment, thus SVM-based DA without bagging could provide sufficient predictivity. The improved DAs in this study could be promising tools for skin sensitization assessment without animal testing.

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever.

The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and wild boars is caused by African swine fever virus (ASFV), can reach 100%. Since the first confirmed ASF outbreak in China on 3 August 2018, 156 ASF outbreaks were detected in 32 provinces. About 1,170,000 pigs were culled in order to halt further spread. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12a-based On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout and the assay displayed no cross-reactivity to other 13 swine viruses including classical swine fever (CSF). CORDS could identify the ASFV DNA target at femtomolar sensitivity in an hour at 37°C, and only requires an incubator. For ease of use, the reagents of CORDS were lyophilized to three tubes and remained the same sensitivity when stored at 4°C for at least 7 days. Thus, CORDS provide a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field-based applications.