#INSPECTR is a #syntheticbiology based #moleculardiagnostics platform developed at #WyssInstitute to benefit the consumers directly with a #low_cost and self-sufficient alternative. The #INSPECTR assay is being dubbed as #NASBACC or NASBA-CRISPR Cleavage kind of assay as it uses the power and affordability of #PaperBasedDetection.
Principle
#INSPECTR is an abbreviation of “Internal Splint-Pairing Expression Cassette Translation Reaction,” consists of a #DNA hybridization-based sensor that can be easily programmed to detect target nucleic acids (DNA or RNA) with single base pair specificity, coupled with a paper-based synthetic gene network that translates the sensor’s detection into a #bioluminescent signal that is easily visualized or captured on instant film. Most importantly, this can be performed at room temperature and does not require any instrumentation, unlike most other available methods.
Protocol
NASBA: Nucleic Acid Sequence Based Amplification (#NASBA) is a typical isothermal RNA amplification process. Here its constituent reverse primer is appended with the trigger sequence of a synthetic toehold switch (sensor H), so that the intermediary #dsDNA products has sensor H.
Cas9 cleavage: In the presence of the appropriate #PAM sequence and guide #RNA target site, the #dsDNA that is synthesized as part of the NASBA reaction undergoes Cas9-mediated cleavage, resulting in a truncated #RNA product that is unable to activate the sensor H toehold switch.In the absence of the PAM sequence, the full-length #RNA product containing the sensor H trigger sequence is generated, allowing for sensor H activation.
Toehold Switch based detection: Toehold switch sensors are programmable synthetic #riboregulators which control the translation of a specific gene in vitro via the binding of a trans-acting trigger #RNA. The switches contain a hairpin structure that blocks gene translation in cis by sequestration of the ribosome binding site (#RBS) and start codon. Upon a switch binding to a complementary trigger #RNA (sensor H here), RBS and start codon is relieved, activating gene translation. To allow for colorimetric detection of trigger #RNA sequences, enzyme #LacZ is regulated, where #LacZ mediates a color change by converting a yellow substrate (chlorophenol red-b-D-galactopyranoside) to a purple product (chlorophenol red).
Merits
Instrument-free assay cutting down cost and logistics for on-field detection!
#Simple to perform and read the results
#FemtoMolar detection limit (#LoD)
Cheap (~$1/ test)
#NASBA reagents are freeze-dry compatible, making it #portable without freezers.
Demerits
Sample preparation requires #heating to 95C, which is a limiting factor for the sensor.
Applications
Molecular diagnostics for detection of a number of #pathogenic diseases.
#StrainDifferentiation of pathogens.
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