Es mostren les entrades ordenades per data per a la consulta crispr diagnostics. Ordena per rellevància Mostra totes les entrades
Es mostren les entrades ordenades per data per a la consulta crispr diagnostics. Ordena per rellevància Mostra totes les entrades

19 de juliol 2023

Canviant la funció de producció de les proves diagnòstiques

 Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics

Durant la pandèmia es va produir una innovació notable a la tecnologia de proves diagnòstiques. Va passar desapercebuda per alguns, però no pels que llegiu aquest blog. Es tracta d'utilitzar CRISPR que inicialment s'ha desenvolupat per a edició genètica, per a la detecció d'àcids nucleïcs en un sol pas, i per tant també de Sars-COV-2. La dificultat d'aplicació que tenia aquella prova, que va rebre el vist-i-plau de la FDA era que el procés no es podia automatitzar, i per tant calia amplificació prèvia. Ara acaba de publicar-se la prova definitiva que pot capgirar moltes coses, i que per tant pot canviar la funció de producció dels laboratoris. La troballa d'enzims termoestables ha estat la qüestió clau per a l'èxit de l'equip de Feng Zhang.

Cal dir que la companyia rival, Mammoth Biosciences, de Jennifer Doudna,  també té en marxa una prova similar: DETECTR BOOST.

I ara ja ha començat el procés de patentar enzims. Tant per una empresa com per l'altre. I això esdevé inadmisible si el que es pretén patentar la natura o variacions sobre la natura. Però ningú se n'està adonant , als USA ja s'ha fet tard, però a Europa encara hi som a temps.

N'estic segur que les patents, una vegada més, limitaran l'accés a aquesta gran innovació.



Cartier-Bresson


PS. Per si voleu saber com funciona DETECTR BOOST

PS. Per tal de comprendre l'abast del que signifiquen les proves diagnòstiques basades en CRISPR el millor és consultar un article de revisió com aquest. I la taula següent conté les dades bàsiques:

Characteristics of reported CRISPR-based diagnostics

From: CRISPR-based diagnostics

Name

Enzyme

Preamplification

Assay timea (min)

Sample preparation

Readout

Applications

LODc (mol l−1)

LODc (copies per ml)3

References

CRISPR type II

NASBACCb

Cas9

NASBA

120–360 (one pot)

Column-based or crude extraction

Colometry

Discrimination between African and American ZIKV

1.0 × 10–15

6.0 × 105

25

CRISPR–Chip

Cas9

15

Column-based

Electrochemical

Detection of gDNA from cell lines and DMD patients

2.3 × 10–15

1.4 × 106

45

CRISDA

Cas9 nickase

SDA

90

Column-based

Fluorescence

Detection of gDNA; breast-cancer-associated SNPs in cell lines

2.5 × 10–19

1.5 × 102

27

FLASH

Cas9

PCR

NS

Column-based

NGS

Detection of gDNA; antimicrobial resistance genes in clinical samples

1.9 × 1018

1.1 × 103

71

CAS-EXPAR

Cas9

EXPAR

60

Chemical (phase separation)

Fluorescence

Sensing of methylated DNA; L. monocytogenes mRNA

8.2 × 10–19

4.9 × 102

91

Cas9nAR

Cas9 nickase

Strand-displacing DNA polymerase

60

Column-based

Fluorescence

Detection of bacteria (S. typhimurium, E. coliM. smegmatisS. erythraea); detection of KRAS SNPs in cell lines

1.7 × 10–19

1.0 × 102

111

CRISPR type V

DETECTR

Cas12a

RPA

10 (RPA) and 60–120 (CRISPR)

Crude extraction

Fluorescence

Detection of HPV16 and HPV18 in human samples

1.0 × 10–18

6.0 × 102

36

Cas14-DETECTR

Cas14 (Cas12f)

PCR

NS (PCR) and 120 (CRISPR)

Crude extraction

Fluorescence

Detection of HERC2 SNPs in human samples

n.s.

6.0 × 103

41

HOLMES

Cas12a

PCR

88 (PCR) and 15 (CRISPR)

Column-based

Fluorescence

SNP discrimination in cell lines and human samples; detection of viruses (PRV, JEV); virus-strain discrimination

1.0 × 10–17

6.6 × 103

37,38

CRISPR-materials

Cas12a

RPA

40 (RPA) and 240 (CRISPR)

Synthetic targets

Fluorescence or μPAD (visual and electronic)

Detection of EBOV synthetic RNA

1.0 × 10–17

6.6 × 103

79,80

CDetection

Cas12b

RPA

10 (RPA) and 60–180 (CRISPR)

Synthetic targets or crude extraction

Fluorescence

Detection of HPV16; human ABO blood genotyping; BRCA1 and TP53 SNPs

1.0 × 10–18

6.0 × 102

112

HOLMESv2

Cas12b

LAMP

40 (LAMP) and 35 (CRISPR) or 120 (one pot)

NS

Fluorescence

SNP discrimination in cell lines; RNA virus detection (JEV); human mRNA and circular RNA detection; DNA methylation

1.0 × 10–17

6.0 × 103

39

E-CRISPR

Cas12a

30–180

Synthetic targets (nucleic acids)

Electrochemical

Detection of viruses (HPV16, PB19) and protein (TGF-ß1)

5.0 × 10–11

3.0 × 1010

77

CRISPR type VI

 –

Cas13

NS

NS

Fluorescence

Detection of human mRNA; detection of bacteriophage λ-RNA

1.0 × 10–12

6.0 × 108

30,31

SHERLOCK

Cas13

NASBA or RPA

132 (NASBA) or 120 (RPA) and 60–180 (CRISPR)

Column-based or crude extraction

Fluorescence

Detection of viruses (ZIKV, DENV) and bacteria (E. coliK. pneumoniaeP. aeruginosaM. tuberculosisS. aureus); discrimination between virus strains; detection of SNPs

2.0 × 10–18

1.2 × 103

32

SHERLOCKv2b

Cas13

RPA

60 (RPA) and 60–180 (CRISPR) or 60–180 (one pot)

Column-based or crude extraction

Fluorescence or lateral flow

Detection of viruses (ZIKV, DENV) and bacteria (P. aeruginosaS. aureus); discrimination between virus strains; detection of SNPs

8.0 × 10–21

4.8

22,34

SHINEb

Cas13

RPA

50 (one pot)

Crude extraction

Fluorescence or lateral flow

Detection of SARS-CoV-2

8.3 × 10–18

5.0 × 103

62

STOPCovidb

Cas12b

LAMP

60 (one pot)

Crude extraction

Fluorescence or lateral flow

Detection of SARS-CoV-2

3.3 × 10–18

2.0 × 103

63

CARMEN

Cas13

PCR or RPA

20 (RPA) and 180 (CRISPR)

Column-based

Fluorescence

Detection of 169 viruses; subtyping of influenza A strains; detection of HIV drug-resistant mutations

9 × 10–19

5.4 × 102

67

APC-Cas

Cas13

Allosteric-probe-initiated amplification with DNA polymerase

110 (APC) and 30 (CRISPR)

None

Fluorescence

Detection of S. enteritidis

One colony-forming unit

92

 

Cas13

<240

Column-based

Electrochemical

Detection of microRNAs (miR-19b and miR-20a)

1 × 10–11

6.0 × 109

46

PECL-CRISPR

Cas13

EXPAR

30 (CRISPR), 30 (phosphorylation of pre-trigger), 30 (EXPAR)

Column-based

Electrochemiluminescence

Detection of microRNAs (miR-17, let‐7 family miRNAs)

1.0 × 10–15

6.0 × 105

78

  1. NS, not specified; APC-Cas, allosteric probe-initiated catalysis and CRISPR-Cas13a system; BRCA1, breast cancer 1 gene; circRNA, circular RNA; Cas9nAR, Cas9 nickase-based amplification reaction; CRISDA, CRISPR–Cas9-triggered nicking endonuclease-mediated strand-displacement amplification; DENV, dengue virus; DMD, Duchenne muscular dystrophy; EBOV, Ebola virus; E. coliEscherichia coliHERC2, HECT and RLD domain containing E3 ubiquitin protein ligase 2 gene; HPV, human papillomavirus; JEV, Japanese encephalitis virus; K. pneumoniaeKlebsiella pneumoniaeKRAS, KRAS proto-oncogene GTPase; M. smegmatisMycobacterium smegmatisM. tuberculosisMycobacterium tuberculosis; PECL, portable electrochemiluminescence chip; P. aeruginosaPseudomonas aeruginosa; PB19, parvovirus B19; PRV, pseudorabies virus; S. erythraeaSaccharopolyspora erythraeaS. aureusStaphylococcus aureusS. enteritidisSalmonella enteritidisS. typhimuriumSalmonella typhimuriumTP53, tumour protein P53 gene.
  2. aAssay time indicates the approximate incubation time most frequently used in the referred study (different assay times can be reported, depending on the intended sensitivity and the readout).
  3. bPOC compatibility indicates whether the entire assay as reported—including sample preparation (that is, crude extraction) and readout—can be performed at POC or in the field with minimal equipment.
  4. cLimits of detection cannot always be directly compared across studies, in particular because some studies did not report how the LOD was determined, or reported the target concentration either in the transport media of the sample or in the final reaction. The LODs shown here reflect the optimal LODs reported. In general, LODs depend on the type of input material (raw or synthetic), type of readout and incubation time.