Pathogen detection Kits:

Karius Digital™ culture kit ™ culture kit (tests for over 1,250 pathogens including bacteria and viruses)

Diagnostic Assay related to the USE of CRISPR effector systems:

CRISPR-Cas12 based detection Assays:

Chiu (“CRISPR-Cas12-based detection of SARS-CoV-2” Nat Biotechnol. 202, 38(7): 870-874) discloses development and validation of a CRISPR-cas12 based assay for detection of SARS-CoV-2 from extracted patient sample RNA, called SARS-CoV-2 DNA Endonuclease targeted CRISPR Trans Reporter (DETECTR). The assay performs simultaneous reverse transcription and isothermal amplificaiton using loop mediated amplificaiton (RT_LAMP) for RNA extracted from nasopharyngeal or oropharyngeal swabs in universal transport media, followed by Cas12 detection of predefined coronavirus squences, after which cleavage of a reporter molecule confirms detection of the virus. 

Cas13-based nucleic acid detection platforms: 

–Specific High Sensitivity Enzymatic Reporter Unclocking (SHERLOCK):

SHERLOCKS combines isothermal amplificaiton using Recombinase Polymerase Amplificaiton (RPA)/12 iwth highly specific Cas13 based detection. Cas13, an RNA guided ribonuclease, provides specificity thorugh CRISPR RNA (crRNA): target pairing, and additional sensitivity due to signal amplifcation by Cas13’s collateral cleavage activity. (Sabeti, “Field-deployable viral diagnostics using CRISPR-Cas13” Science, 2018, 360(6387).

Zhang (US 11,633,732) disclsoes a lateral flow diagnostic device referred to as “SHERLOCK” systems which includes a substrateand a first end which includes a loading porition. The first end may also include a rirst region loaded with a detectable ligand, a CRISPR effector system, a detection construct, a first test band that includes a biotin ligand and a second test bank that includes a capture molecule for the detectable ligand. The detection construct can include an RNA oligonucleotide that includes a first molecule such as FITC on a first end and a second molecule such as FAM on a second end. Contacting the sample loading porition with a sample causes the sample to flow from the sample loading porition of the substrate towards the first and second capture regions. In specific embodiments, the first capture region may include a first binding agent that specifically binds the first molecule of the reporter construct. The first bnding agent may be an antibody, such as an anti-FITC antibody which is fixed or immbilied to the frist capture region. The device may further include a second capture region which is located towards the opposite end of the lateral flow substrant form the first binding region. The second capture region may include a second bnding agent that specifically binds the second molecule of the reporte construct such as an antibody such as an anti-biotin antibody which is fixed or immobilized to the second capture region. If a target molecule is present in a sample, the corresponding guide molecule will guid the CRSIPR Cas/guide complex to the target molecule by hydrizing with the target molecule, thereby triggering the CRISPR effector protein’s nuclease activity. This activated CRISPR effector protein will cleave both the target molecule and then non-specifically cleave the linker porition of the RNA construct. In otehr words, if target molecule(s) are present in the sample, the CRISP effector protein collateral effect is activated. An activated CRISPR effector protein comes into contact with the bound reporter construct, the reporte construct is cleaved, relasing the seocnd molecule to flow further down the lateral flow substrate towards the second binding region. The released seocnd molecule is then captured at the second capture region by binding to the second binding agent, where additional detection agent may also accumulate by binding to the second molecule. Adcording, if the target molecule(s) is not present int he sample, a detectable signal will appear at the first capture region, and if the target molecule(s) is present in the sample, a detectable signal will appear at the location of the second capture region. Oligonucleotide linkers haivng molecuels on either end may include DNA if the CRISPR effector potein ahs DNA colateral activity or RNA if the CRISPR reffecotr protoein has RNA collateral activity. Oligonucleotide linkers may be single or double stranded and in certain embodimens they could contain both RNA and DNA regions. For example, a lateral flow strip allows for RNAse (e.g., Cas13a) detection by colar. The RNA reporter is modified to have a first molecule such as FITC attached to the 5’end and a second molecule such as biotin attached to the 3′ end of vice versa. The lateral flow strip is designed to have two capture lines with anti-first molecule such as anti-FITC antibodies hydridized at the first line and anti-seocnd molecule such as anti-biotin antibodies at the seocnd downstream line. As the SHERLOCK reaciton flows down the strip, uncleaved reported will bind to the anti-first molecule antibodies at the first capture line, while cleaved reported will liberate the seoond molecule and allow second molecule binding at the second cpature line. Second mocluel sadnwich antibodies such as conjugated to nanoparticles such as gold nanoparticles will bind any second molecule at the first or second line and result in a strong readout/signal (e.g., color). 

SHERLOCK can identify SNPs by placing a synthetic mismatch in the crRNA near the SNP, testing each target with an acestral-specific and derived specific crRNA. Sabeti, “Field-deployable viral diagnostics using CRISPR-Cas13” Science, 2018, 360(6387)

–Heating Unextracted Diagnostic Samples to Obliterate Nucleases (HUDSON): 

(Sabeti, “Field-deployable viral diagnostics using CRISPR-Cas13” Science, 2018, 360(6387) disclose detection of viral nucleic acid directly from bodily fluids via HUDSON, a method to lyse viral particles and inactivate the high levels of RNases found in boidly fluids uisng heat and chemical reduction. HUDSON treated urine or soliva could be direclty added to RPA reactions without delution or purification. HUDSON and SHERLOCK enabled sensitive detection of free ZIKV nucleic acid spiked into urine, whole blood, plasma, serum, or saliva. HUDSON is a protocol that paris with SHERLOCK for viral detection direclty from bodily fluids, enalbing instrument free DENV detection direclty form patient samples in <2 hours. Sabeti showed that SHERLOCK can distinguish the 4 DENV seriotpyes as well as region specific strains of ZIKV. 

–Multiplexing: 

(Zhang, “Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6” Science 208, 360(6387): 439-444) discloses identifying possible candidate enzymes compatible with multiplexing by characterizing three members of the CRISPR-Cas13a family and 14 members of the CRISPR-Cas13b family. They profiled cleavage preferences on homopolymer reporters and found that most ortholgos prefered wither uridei, or a combaintion of bases, or adenine and clevage could be improved with buffer and crRNA design optimization. Using these unique clevage preferences, they were able to detect synthetic Zika virus ssRNA in an HEX channel and synthetic Dengue Virus (DENV) ssRNA in a FAM channel in the same reaction. 

—-Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN): See YouTube

Sabeti “”Massively multiplexed nucleic acid detection with Cas13” Nature, 582, June 2020) discloses development of CARMEN, a platform for scalable, mltiplxed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR based nucleic aciddetection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combaintion fo CARMEN and Cas13 detection (CARMEN-Cas13) enablesrobust testing of more than 4,500 crRNA target pairs on a single array. Using CARMEN-Cas13, they developed a multiplexed assay that simultaneously differentiates all 169 human assocaited virsues with at least 10 published genome sequenes. The inputs of CARMEN-Cas13 are samples that have been amplified by PCR or recombinase polymerase amplificaiton (RPA) and Cas13-detection mixes, which coontain Cas13, a sequence specific CRISPR RNA (crRNA) and a cleavage reporter. Each amplified sample or detection mix is prepared in a conventional microtitre plate and combined with a distinct, solution based fluorescent colour code that serves as an optical identifier. Each colour coded solution is emulsified in fluorous oil to yield 1 nl droplets. Once emulsified droplets form all samples and detection mixes are pooled into a single tube and in one pipetting step are loaded into a microwell array chip moulded form PDMS.