Existing methods of detecting foreign
genes and their expression
products from genetically modified organisms (GMOs) suffer from the
requirement of professional equipment and skillful operators. The
same problem stays for the CRISPR-Cas12a system, although it has been
emerging as a powerful tool for nucleic acid detection due to its
remarkable sensitivity and specificity. In this report, a portable
platform for the visible detection of GMOs based on CRISPR-Cas12a
was established, which relies on a color change of gold nanorods (GNRs)
caused by the invertase-glucose oxidase cascade reaction and the Fenton
reaction for signal readout. A nopaline synthase (NOS) terminator
was employed as a model target commonly existing in foreign genes
of GMOs. With the help of recombinase-aided amplification, this platform
achieved comparable sensitivity of DNA targets (1 aM) with that of
a fluorescence reporting assay. As low as 0.1 wt % genetically modified
(GM) content in Bt-11 maize was visually observed by unaided eyes,
and the semiquantitation of GM ingredients can be obtained within
the range of 0.1 to 40 wt % through the absorption measurement of
GNRs. Furthermore, five real samples were tested by our method, and
the results indicated that the GM ingredient percentages of GMO samples
were 2.24 and 24.08 wt %, respectively, while the other three samples
were GMO-free. With the advantages of a simple procedure, no need
for large or professional instruments, high sensitivity, and selectivity,
this platform is expected to provide reasonable technical support
for the safe supervision of GMOs.
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has spread rapidly throughout the whole world and caused significant difficulties in the prevention and control of the epidemic. In this case, several detection methods have been established based on nucleic acid diagnostic techniques and immunoassays to achieve sensitive and specific detection of SARS‐CoV‐2. However, most methods are still largely dependent on professional instruments, highly trained operators, and centralized laboratories. These limitations gravely diminish their practicality and portability. Herein, a clustered regularly interspaced short palindromic repeats (CRISPR) Cas12a based assay was developed for portable, rapid and sensitive of SARS‐CoV‐2. In this assay, samples were quickly pretreated and amplified by reverse transcription recombinase‐aided amplification under mild conditions. Then, by combining the CRISPR Cas12a system and a glucose‐producing reaction, the signal of the virus was converted to that of glucose, which can be quantitatively read by a personal glucose meter in a few seconds. Nucleocapsid protein gene was tested as a model target, and the sensitivity for quantitative detection was as low as 10 copies/μl, which basically meet the needs of clinical diagnosis. In addition, with the advantages of lower material cost, shorter detection time, and no requirement for professional instrument in comparison with quantitative reverse transcription‐polymerase chain reaction, this assay is expected to provide a powerful technical support for the early diagnosis and intervention during epidemic prevention and control.
Invasive
fungi (IF) have become a significant problem affecting
human health. However, the culture-based assay of IF, known as the
most commonly used clinical diagnostic method, suffers from time consumption,
complicated operation, and the requirement of trained operators, which
may cause the delay diagnosis of the disease. In this report, a microfluidic
ruler-readout and CRISPR Cas12a-responded hydrogel-integrated paper-based
analytical device (μReaCH-PAD) was established for visible and
quantitative point-of-care testing of IF. Using the genus-conserved
fragments of 18s rRNA as the detection target, this platform relied
on a CRISPR Cas12a system for target recognition, a DNA hydrogel coupled
with a cascade of enzymatic reactions for signal amplification and
transduction, and paper-based microfluidic chips for visual quantitative
readout by naked eyes. The 18s rRNA fragments of Candida or Aspergillus were employed as a model target
and introduced with PAM sites for Cas12a-recognition during reverse
transcription recombinase-aided amplification. Using μReaCH-PAD,
as low as 10 CFU/mL Candida and Aspergillus were visually identified by unaided eyes. The calculated detection
limits were 4.90 and 4.13 CFU/mL (in 1 mL samples), respectively.
The quantitative detection results can be obtained in the range from
10 to 104 CFU/mL with reasonable specificity and accuracy
compared with qRT-PCR. Furthermore, μReaCH-PAD can analyze complex
biological samples by Candida, Aspergillus, and Cryptococcus detection systems and identify
specific genera of different IF by naked eyes, indicating a good agreement
with the culture-based assay and the advantages over G-testing and
GM-testing systems. With the benefits of high sensitivity, selectivity,
quantitative readout, low cost, and ease of operation, μReaCH-PAD
is expected to provide a portable detection tool of IF in resource-limited
settings by untrained personnel and technical support for early diagnosis.
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