Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology

Abstract

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10−4 single cysts and single females, 4−3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.

Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system

“…The integration of the CRISPR-Cas12a and nucleic acid amplification techniques has been proved by several works. 22,60,63–65 These results demonstrated that our nanopore sensor using a DNA tetrahedron as a signal transducer based on the CRISPR-Cas12a conversion mechanism could detect targets specifically, which we believe could become a promising alternative to biomolecular detection.…”

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13

“… 33 A CRISPR-Cas12 system may be used to detect the genome of these organisms as well, as already reported in some pioneer studies. 34 − 36 Furthermore, because of the exquisite sequence recognition ability of these systems, the genetic diversity of a given pathogen might be detected (at least some variants). Continuous monitoring of different pathogens in the field should help to implement better control strategies incorporating ecological concepts.…”

Potential of the CRISPR‐Cas system for improved parasite diagnosis

“…Apparently, it is critical and necessary to develop a rapid and field-friendly diagnosis tool for better prevention and control this disease. Yao et al [74] combined RPA with CRISPR-Cas12a technology to successfully detect plant samples infected with H. schachtii within 1 hour by fluorescence. The sensitivity of the system developed was greater than electrophoresis and was similar to that of an RPA/lateral flow dipstick procedure reported in the same study.…”

“…Supported by the ever expanding numbers of pre-clinical and clinical studies which have shown the utility of CRISPR-Cas12/13-based diagnosis for viral diseases (especially SARS-CoV-2) and cancer, the CRISPR-mediated approach has been extended for the ultrasensitive detection of the single-celled parasites including Cryptosporidium parvum, [34,35] Enterocytozoon hepatopenaei, [36] malaria, [33,73] and a plant-parasitic nematode -Heterodera schachtii. [74]…”