Cheap TE Buffer DNA Extraction Powers Rapid CRISPR Test for Babesia Parasites

A team of researchers in Thailand has demonstrated that a remarkably simple DNA extraction method—nothing more than Tris–EDTA (TE) buffer—can replace expensive commercial kits in a rapid CRISPR-based diagnostic test for Babesia parasites. The work, published in Molecular Biology Reports, describes a streamlined workflow that combines TE buffer extraction with recombinase polymerase amplification (RPA) and CRISPR–Cas12a detection, offering a practical route to near point-of-care diagnosis of a tick-borne pathogen that infects domestic animals, wildlife, and humans alike.

Babesia species are single-celled parasites that invade red blood cells and are transmitted by ticks. In cattle, infections such as babesiosis cause fever, anemia, and significant economic losses, while in dogs the disease can be life-threatening. Human babesiosis, though less common, can be severe in elderly or immunocompromised patients. Because symptoms often overlap with other febrile illnesses, accurate molecular detection is essential—but conventional PCR-based diagnostics typically require laboratory infrastructure, thermal cyclers, and costly reagents that are out of reach in many field and rural settings.

The research team, led by Suphaporn Paenkaew and Kittisak Buddhachat at Naresuan University, set out to address one of the biggest bottlenecks in molecular diagnostics performed outside a laboratory: getting usable DNA out of a blood sample quickly and cheaply. The group evaluated five different blood DNA extraction approaches: a commercial DNeasy kit, simple boiling, TE buffer extraction, a methanol-based method, and Chelex 100 resin. Each extract was then fed into an RPA/CRISPR-Cas12a assay targeting the cytochrome c oxidase subunit I (COI) gene of Babesia.

Three of the five methods—the commercial kit, boiling, and TE buffer extraction—produced positive results in the CRISPR assay. The researchers then focused on the two simplest candidates, TE buffer extraction and boiling, and optimized the assay using Babesia-specific primers designed against the parasite’s 18S ribosomal RNA gene. When the performance of all extraction methods was compared head-to-head, they proved comparable to the commercial kit, but the TE buffer approach stood out for its simplicity and speed, requiring essentially no specialized equipment beyond a pipette and a source of the buffer itself.

The detection chemistry behind the assay is what makes the workflow so powerful. Recombinase polymerase amplification is an isothermal technique that copies DNA at a constant temperature—typically between body temperature and 42 degrees Celsius—without the cycling demanded by conventional PCR. In this study, the team optimized RPA conditions across a range from 25 to 45 degrees Celsius and incubation times from zero to 30 minutes, establishing conditions that work rapidly with minimal instrumentation. Once amplified, the target DNA triggers CRISPR–Cas12a, an enzyme that, upon binding its guide RNA-directed target sequence, unleashes indiscriminate cutting of nearby single-stranded DNA reporters, producing a detectable fluorescent signal.

Specificity is a critical concern for any diagnostic, and the assay passed that test. The RPA/CRISPR-Cas12a system specifically detected Babesia species including Babesia vogeli, Babesia gibsoni, and Babesia ovata—pathogens responsible for canine and bovine babesiosis in tropical and subtropical regions. The guide RNA was designed against a conserved region within the 18S rRNA gene amplification segment, with the protospacer adjacent motif and primer binding regions verified through multiple sequence alignments of Babesia sequences.

Sensitivity measurements revealed a trade-off worth noting. Using DNA extracted with TE buffer, the limit of detection was 10,000 copies per reaction, whereas the commercial kit achieved 1,000 copies per reaction—a tenfold difference in analytical sensitivity. The authors attribute this gap to the relative purity of kit-extracted DNA, which undergoes binding, washing, and elution steps that remove inhibitors. Even so, the TE-based workflow remained fully capable of detecting infections in clinical samples, suggesting that for field screening the modest sensitivity loss is an acceptable price for a dramatic reduction in cost and complexity.

The real-world validation came from clinical blood samples: seven bovine and eight canine specimens were tested using both the TE buffer extraction and the commercial kit, each followed by the RPA/CRISPR-Cas12a assay. The results showed complete concordance between the two extraction approaches, with six positive samples detected. The canine samples were additionally characterized by qPCR high-resolution melting analysis and conventional PCR with sequencing, confirming that the positives reflected genuine Babesia infections rather than assay artifacts.

The implications extend beyond veterinary clinics. The same research group has previously deployed RPA/CRISPR-Cas12a assays for other tick-borne pathogens, including Ehrlichia canis, Anaplasma platys, and Hepatozoon canis in dogs, and for Babesia vogeli specifically. Building a reliable, kit-free extraction step into that pipeline removes one of the last laboratory-dependent components. In regions where babesiosis is endemic and diagnostic laboratories are scarce, a workflow that needs only TE buffer, isothermal amplification reagents, and a CRISPR detection mix could be run in veterinary practices, farms, or mobile clinics.

The study, funded by the National Research Council of Thailand and Naresuan University, positions TE buffer extraction as a pragmatic bridge between sophisticated molecular diagnostics and the resource-limited settings that need them most. While the tenfold sensitivity gap relative to commercial kits may matter for very low-level infections, the complete agreement on clinical samples indicates that the simple method is fit for purpose. As CRISPR-based diagnostics continue to mature, studies like this one underscore that the humblest reagents—buffer, primers, and a guide RNA—can deliver laboratory-quality answers where they are needed fastest.

Subject of Research: A low-cost TE buffer DNA extraction method combined with RPA/CRISPR-Cas12a for rapid point-of-care detection of Babesia parasites

Article Title: A cost-effective and rapid TE-based DNA extraction for near point-of-care detection of Babesia spp. by RPA/CRISPR-Cas12a

Article References: Paenkaew, S., Sinthao, O., Thapthim, T., Mahakkaprateep, S., Cheukaw, A., Keawmanee, S., Euppayo, T., Tungtrakanpoung, R., Teapunvong, W., Nganvongpanit, K., & Buddhachat, K. (2026). A cost-effective and rapid TE-based DNA extraction for near point-of-care detection of Babesia spp. by RPA/CRISPR-Cas12a. Molecular Biology Reports, 53(1), Article 1545. https://doi.org/10.1007/s11033-026-12665-2

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12665-2

Keywords: Babesia, DNA extraction, TE buffer, CRISPR-Cas12a, recombinase polymerase amplification, point-of-care diagnostics, babesiosis, 18S rRNA, veterinary parasitology, tick-borne disease, molecular detection, isothermal amplification