Technology for Early HLB Diagnosis

Ernie NeffTechnology

Citrus trees infected with huanglongbing disease often display asymmetrical yellowing of the leaves and leaf veins — referred to as “blotchy mottle.”
Photo by David Bartels, USDA Animal and Plant Health Inspection Service

Penn State and U.S. Department of Agriculture (USDA) scientists have used cutting-edge CRISPR/Cas technology to develop a diagnostic test that could enable early diagnosis of huanglongbing (HLB).

In a study newly published in the journal Phytopathology, the researchers demonstrated that the new test can detect the presence of the HLB causal agent — the bacterium Candidatus Liberibacter asiaticus (CLas) — at a sensitivity level 100 to 1,000 times greater than a commonly used diagnostic test, quantitative polymerase chain reaction (qPCR).

Early detection of the pathogen is crucial because infected trees can act as an HLB reservoir for months or years before showing visible symptoms. Researchers deployed CRISPR/Cas to address the need for early diagnosis, stated study co-author Yinong Yang. Yang is a professor of plant pathology in Penn State’s College of Agricultural Sciences.

“So far, various technologies, such as canine olfactory detection and qPCR, have been used to diagnose and confirm CLas infection,” said Yang. “But these tools are often inadequate for early detection of CLas in asymptomatic tissue — where low amounts of bacteria are present — and are unsuitable for high-throughput diagnosis in the field.”

CRISPR stands for clustered regularly interspaced short palindromic repeats. The technology can modify an organism’s genome by precisely delivering a DNA-cutting enzyme — Cas — to a targeted region of DNA. The resulting modification can delete or replace specific DNA pieces, thereby promoting or disabling certain traits.

The researchers adapted the CRISPR/Cas system to develop an assay based on a platform known as DETECTR, which stands for DNA endonuclease-targeted CRISPR trans reporter. In this assay, a Cas variant called Cas12a is mixed with CRISPR RNA designed to seek out CLas DNA, and a synthetic molecule known as a fluorescent reporter oligonucleotide. If CLas DNA is present, the Cas12a cleaves both the pathogen’s DNA and the reporter oligonucleotide, enabling the generation of a fluorescent signal for detection.

The researchers report that this assay was highly sensitive and specific in detecting CLas DNA, using either a microplate reader for fluorescence readouts or a lateral flow strip for a visual result indicating the presence of a target sequence — similar in concept to a home pregnancy test.

“The assay successfully detected and confirmed the presence of CLas nucleic acids in infected samples collected from Florida,” said study lead author Matthew Wheatley. Wheatley is a postdoctoral scholar in plant pathology and environmental microbiology at Penn State.

Yong-Ping Duan, research plant pathologist at the USDA Agricultural Research Service’s U.S. Horticultural Research Laboratory in Fort Pierce, Florida, was also a member of the research team.

Penn State’s College of Agricultural Sciences, USDA’s National Institute of Food and Agriculture, and the USDA Agricultural Research Service supported the work.

Source: Penn State

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