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group_6_ta_sansi_xing_diagnostics_of_sars-cov-2 [2021/02/02 00:45] nimojant [CRISPR-Cas9] |
group_6_ta_sansi_xing_diagnostics_of_sars-cov-2 [2021/02/27 20:09] (current) dhalip1 [Diagnostics of SARS-Cov-2] |
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| ===== What is SARS-CoV-2? ===== | ===== What is SARS-CoV-2? ===== | ||
| - | <box 25% round right | > | + | <box 30% round right | > |
| - | {{ ::23311_lores.jpg?300 |}} | + | {{ ::23311_lores.jpg?275 |}} |
| </box|Figure 1: Illustration of SARS-CoV-2. Adapted from Eckert & Higgins (2020).> | </box|Figure 1: Illustration of SARS-CoV-2. Adapted from Eckert & Higgins (2020).> | ||
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| </box| Figure 8: SARS-CoV-2 DETECTR workflow. Conventional RNA extraction can be used as an input to DETECTR which is then visualized by a fluorescent reader or lateral flow strip (Broughton et al., 2020).> | </box| Figure 8: SARS-CoV-2 DETECTR workflow. Conventional RNA extraction can be used as an input to DETECTR which is then visualized by a fluorescent reader or lateral flow strip (Broughton et al., 2020).> | ||
| - | CRISPR–Cas12-based assay for detection of SARS-CoV-2 from extracted sample RNA. This detection method is called SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR). This assay performs simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP), followed by Cas12 detection of predefined coronavirus sequences. Then a reporter molecule is cleaved to confirm the detection of the virus. The extracted RNA from a sample is placed in the DETECTR system where LAMP preamplification and Cas12-based detection for envelope (E) gene, nucleoprotein (N) gene and RNase Polymerase occurs, which is visualized by a fluorescent reader or lateral flow strip | + | CRISPR–Cas12-based assays can be for the detection of SARS-CoV-2 from extracted sample RNA. One detection method using this type of assay is called SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) (Broughton et al., 2020). In the first step of this method, the samples are collected from the patients. The most general forms of the SARS- CoV-2 samples are nasopharyngeal and oropharyngeal swabs (Nouri et al., 2021). After sample collection, viral RNAs need to be extracted from the raw sample. |
| - | Primers that target the envelope (E) and nucleoprotein (N) genes of SARS-CoV-2 were used by this method and are similar to the ones used in the WHO assay and US CDC assay are utilized with slight modifications to be optimally used in the DETECTR system. The N1 and N3 regions used by the US CDC assay weren’t used, as these regions did not have compatible protospacer adjacent motif sites for the Cas12 guide RNAs (gRNAs) used to detect 3 sars like coronaviruses in the E gene and only SARS-CoV-2 in the N gene. | + | RNA isolation procedures typically involve three general steps: lysis, separation of RNA from other macromolecules such as DNA, proteins, and lipids, followed by RNA elution (Nouri et al., 2021). This isolation can be performed by magnetic bead purification, spin column isolation, or organic extraction which is considered the best method for this situation. Once isolation is completed, the isolated sample is amplified using methods such as RT-PCR or RT-LAMP to boost the amount of RNA past the limit of detection (Nouri et al., 2021). |
| + | Once simultaneous reverse transcription and isothermal amplification using RT–LAMP is performed, the sample is placed in the DETECTR system where Cas12 detection of predefined coronavirus sequences occurs (Broughton et al., 2020). Then a reporter molecule is cleaved to confirm the detection of the virus (Broughton et al., 2020). The extracted RNA from a sample is placed in the DETECTR system where LAMP preamplification and Cas12-based detection for envelope (E) gene, nucleoprotein (N) gene and RNase Polymerase occurs, which is visualized by a fluorescent reader or lateral flow strip (Broughton et al., 2020). | ||
| + | |||
| + | Primers that target the envelope (E) and nucleoprotein (N) genes of SARS-CoV-2 were used by this method and are similar to the ones used in the WHO assay and US CDC assay are utilized with slight modifications to be optimally used in the DETECTR system (Broughton et al., 2020). The N1 and N3 regions used by the US CDC assay weren’t used, as these regions did not have compatible protospacer adjacent motif sites for the Cas12 guide RNAs (gRNAs) used to detect 3 sars like coronaviruses in the E gene and only SARS-CoV-2 in the N gene (Broughton et al., 2020). | ||
| ==== Advantages ==== | ==== Advantages ==== | ||
| - | jkjkjkfjk | + | A key advantage of the DETECTR system over the CDC qRT–PCR assay is that the DETECTR system is capable of getting its result at a rate that is 400% faster than the qRT-PCR assay (Broughton et al., 2020). Other key advantages of the DETECTR system over the CDC qRT–PCR assay include isothermal signal amplification without needing thermocycling, rapid turnaround time, single nucleotide target specificity, integration with accessible and easy-to-use reporting formats such as lateral flow strips, and no requirement for complex laboratory infrastructure (Broughton et al., 2020). |
| + | Using synthetically derived SARS-CoV-2 RNA, DETECTR is also able to distinguish SARS-CoV-2 with no cross-reactivity to other strains of coronavirus when the N gene gRNA (Broughton et al., 2020). | ||
| ==== Limitations ==== | ==== Limitations ==== | ||
| - | djkfjkdjfkj | + | When compared to other assays, the estimated limit of detection for the CDC qRT–PCR assay tested by the California Department of Public Health is 1 copy per µl reaction, versus 10 copies per µl reaction for the DETECTR assay making it more likely for this system to produce a false negative (Broughton et al., 2020). |
| ===== High Throughput Assay (P-Best) ===== | ===== High Throughput Assay (P-Best) ===== | ||
| <box 30% round right| > | <box 30% round right| > | ||
| - | {{ ::p-best.jpg?200 |}} | + | {{ ::p-best.jpg?300 |}} |
| </box| Figure 9: P-BEST design and detection results obtained for a set of 384 samples with a carrier rate of ~1% (Shental et al., 2020). > | </box| Figure 9: P-BEST design and detection results obtained for a set of 384 samples with a carrier rate of ~1% (Shental et al., 2020). > | ||
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| ==== Limitations ==== | ==== Limitations ==== | ||
| - | sfdfd | + | The current implementation of P-BEST is designed for a carrier rate of about 1% (Shental et al., 2020). When the carrier rate of a population is higher than this ideal value, the efficiency of the system changes and can lead to errors (Shental et al., 2020). At higher carrier rates too many pools would be PCR positive, and the number of samples identified by the method will be much larger than the number of expected carriers. This will prevent direct identification of the actual positive carriers in a single testing round and the user will have to retest the samples from the positive pools either individually or by using an alternate pooling design (Shental et al., 2020). |
| ===== Presentation Slides ===== | ===== Presentation Slides ===== | ||
| - | {{:coronavirus.pdf|}} | + | {{:diagnostics_of_sars-cov-2.pdf|}} |
| ===== References ===== | ===== References ===== | ||
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