PMID- 35882427
OWN - NLM
STAT- MEDLINE
DCOM- 20220928
LR  - 20220928
IS  - 1365-2672 (Electronic)
IS  - 1364-5072 (Linking)
VI  - 133
IP  - 4
DP  - 2022 Oct
TI  - An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of 
      SARS-CoV-2.
PG  - 2668-2677
LID - 10.1111/jam.15737 [doi]
AB  - AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is 
      sensitive, but high instrumentation cost makes it difficult for use outside 
      routine clinical setup. This has necessitated the development of alternative 
      methods such as CRISPR-based DETECTR method which uses lateral flow technology. 
      Although accurate and sensitive, this method is limited by complex steps and 
      recurrent cost of high-quality lateral flow strips. The main goal of this study 
      was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable 
      and field-deployable system to reduce the recurring consumable cost. METHODS AND 
      RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase 
      P (internal control) were reverse transcribed (RT) and amplified by loop-mediated 
      isothermal amplification (LAMP). The amplified products were detected by a 
      Cas12a-based trans-cleavage reaction that generated a fluorescent signal which 
      could be easily visualized by naked eye. Detection of internal control, RNase P 
      gene was improved and optimized by redesigning RT-LAMP primers. A number of steps 
      were reduced by combining the reagents related to the detection of Cas12a 
      trans-cleavage reaction into a single ready-to-use mix. A portable, 
      cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the 
      assay and visualize the outcome. The method and instrument were validated using 
      both contrived and patient samples. CONCLUSIONS: The simplified CRISPR-based 
      SARS-CoV-2 detection and instrument developed in this study, along with improved 
      design for internal control detection allows for easier, more definitive viral 
      detection requiring only reagents, consumables and the battery operable 
      CRISPR-CUBE. SIGNIFICANCE AND IMPACT OF STUDY: Significant improvement in Cas12 
      method, coupled with simple visualization of end point makes the method and 
      instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, 
      without any recurrent cost for the lateral flow strips which is used in other POC 
      methods.
CI  - (c) 2022 Society for Applied Microbiology.
FAU - Misra, Chitra S
AU  - Misra CS
AD  - Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, 
      Mumbai, Maharashtra, India.
FAU - Rangu, Shyam S
AU  - Rangu SS
AD  - Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, 
      Mumbai, Maharashtra, India.
FAU - Phulsundar, Ravindra D
AU  - Phulsundar RD
AD  - Electromagnetic Application and Instrumentation Division, Bhabha Atomic Research 
      Centre, Mumbai, Maharashtra, India.
FAU - Bindal, Gargi
AU  - Bindal G
AD  - Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, 
      Mumbai, Maharashtra, India.
AD  - Homi Bhabha National Institute, Mumbai, Maharashtra, India.
FAU - Singh, Mandeep
AU  - Singh M
AD  - Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, 
      Mumbai, Maharashtra, India.
FAU - Shashidhar, Ravindranath
AU  - Shashidhar R
AD  - Homi Bhabha National Institute, Mumbai, Maharashtra, India.
AD  - Food Technology Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, 
      India.
FAU - Saha, Tushar K
AU  - Saha TK
AD  - Electromagnetic Application and Instrumentation Division, Bhabha Atomic Research 
      Centre, Mumbai, Maharashtra, India.
FAU - Rao, Akkipeddi V S S N
AU  - Rao AVSSN
AD  - Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, 
      Mumbai, Maharashtra, India.
FAU - Rath, Devashish
AU  - Rath D
AUID- ORCID: 0000-0002-8204-8440
AD  - Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, 
      Mumbai, Maharashtra, India.
AD  - Homi Bhabha National Institute, Mumbai, Maharashtra, India.
LA  - eng
PT  - Journal Article
DEP - 20220803
PL  - England
TA  - J Appl Microbiol
JT  - Journal of applied microbiology
JID - 9706280
RN  - EC 3.1.26.5 (Ribonuclease P)
SB  - IM
MH  - *COVID-19/diagnosis
MH  - CRISPR-Cas Systems
MH  - Humans
MH  - Nucleic Acid Amplification Techniques/methods
MH  - Ribonuclease P/genetics
MH  - *SARS-CoV-2/genetics
EDAT- 2022/07/27 06:00
MHDA- 2022/09/28 06:00
CRDT- 2022/07/26 20:52
PHST- 2022/07/21 00:00 [revised]
PHST- 2022/05/25 00:00 [received]
PHST- 2022/07/22 00:00 [accepted]
PHST- 2022/07/27 06:00 [pubmed]
PHST- 2022/09/28 06:00 [medline]
PHST- 2022/07/26 20:52 [entrez]
AID - 10.1111/jam.15737 [doi]
PST - ppublish
SO  - J Appl Microbiol. 2022 Oct;133(4):2668-2677. doi: 10.1111/jam.15737. Epub 2022 
      Aug 3.