CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples

Abstract

Background Cryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity. Methods By integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor. Results Our ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa. Conclusions This study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment. Graphic Abstract

An Update on Zoonotic Cryptosporidium Species and Genotypes in Humans

“…For example, in the HOLMES (one-HOur Low-cost Multipurpose highly Efficient System) detection platform, if a target DNA is present, a Cas protein called Cas12a forms a complex with the guide CRISPR RNA, which then binds the target DNA and cleaves a non-target ssDNA reporter in the system, resulting in a fluorescent signal [ 233 ]. A lateral flow biosensor has been developed which incorporated isothermal amplification of Cryptosporidium DNA with this CRISPR detection method to identify C. parvum sequences belonging to the gp60 IId subtype family (from both humans and cattle), with high sensitivity and specificity [ 234 ]. This type of system has the potential to be employed in field situations to rapidly identify zoonotic subtypes and transmission hot-spots for more targeted analysis.…”

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

“…Supported by automated microfluidic mixing, an approach for Ebola virus detection was established using Cas13a and achieved an LoD of 20 pfu/ ml (5.45 × 10 7 copies/ml) of purified Ebola RNA within 5 min (Qin et al, 2019). Cas12a-based biosensing was also developed for the detection of a variety of pathogenic microorganisms, such as Listeria monocytogenes , Cryptosporidium parvum (Yu et al, 2021), Salmonella (Ma et al, 2021), Helicobacter pylori (Qiu et al, 2021), Yersinia pestis (You et al, 2021), E. coli, and Staphylococcus aureus (Bonini et al, 2021). Coupling with a reversible valve-assisted chip, sample preparation, Cas12a reactions, and LAMP was integrated and controlled precisely to perform the detection of V. parahaemolyticus, achieving an LoD of 30 copies/reaction within 50 min (Wu et al, 2021b).…”

“…Zhao et al employed DNA-AuNPs to establish the signal transduction between trans-cleavage of CRISPR/Cas12a and protein analytes and showed a quantitative level of attomolar, 1,000-fold more sensitive and 15-fold wider detection range than traditional ELISA (Zhao Q. et al, 2021). CRISPR-based electrochemiluminescence biosensors have been explored for the detection of enzymes like polynucleotide kinase/ phosphatase (Wang et al, 2020) and alkaline phosphatase (Wang et al, 2021d), and signal factors like endogenous chemokine (White et al, 2021) and sialic acid-binding immunoglobulin-like lectins (Zhang K. et al, 2021). Similar to aptamers, DNAzymes or DNA ligations were also designed to introduce CRISPR/Cas12a biosensor for non-nucleic acid targets, for example, testing melamine (Qiao et al, 2021), metal ion Na + (Li C.-Y.…”

“…Considering the device limitation of PCR methods, portable and isothermal amplification methods are preferred adoptions for preamplification and showed great potential with the capabilities to perform under easily controlled conditions, even at physiological temperature (Vincent et al, 2004;Piepenburg et al, 2006;Tomita et al, 2008). Moreover, with the advances of aptamers used in the CRISPR systems, ultrasensitive protein detection and protein/small molecular interaction identification can also be facilitated (Wang et al, 2020;Zhang K. et al, 2021;Wang et al, 2021d;White et al, 2021) (e.g., alkaline phosphatase, polynucleotide kinase/ phosphatase, and chemokine).…”

RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA)

“…[ 38 ] and Cryptosporidium spp. [ 39 ]. However, to date, there has been no report of the use of CRISPR-Cas system for the detection of T. gondii yet.…”