CRISPR diagnostic verifies that meat is halal

Food scams targeting the halal consumer are growing with global Muslim populations. Halal meat should be pork-free and slaughtered in accordance with Islamic law, but sometimes manufacturers slip in pork and cheaper cuts of meat and sell it for the higher halal price. In summer 2020, food authorities in Thailand investigated a widespread scam of pork coated in ox blood sold as beef. To help track halal food fraud, researchers in China propose the first CRISPR/Cas-based test for pork that’s simpler, less expensive, and less prone to contamination than current genetic tests. (J. Agric. Food Chem. 2021, DOI: 10.1021/acs.jafc.1c03078).

In the US, if “halal” is written on a meat product, it means the manufacturer went through halal certification and usually sends a yearly meat sample to a laboratory for pork testing. The favored workhorse test for food authentication is quantitative polymerase chain reaction (qPCR), which amplifies and measures meat DNA. Although protein tests exist, proteins denature with processing and heat. DNA detection is more reliable, especially for cooked foods, says Ruijie Deng, an analytical chemist at Sichuan University who led the team that developed the diagnostic.

Yet qPCR requires expensive lab equipment and trained operators. A cheaper, easier DNA test would open the door to far more frequent testing, Deng says, for example, whenever a meat production line gets tweaked. Research groups and companies are evaluating CRISPR/Cas technology as the next generation in nucleic acid detection that could replace PCR.

Deng’s CRISPR test targets the gene in pig mitochondria that encodes cytochrome b, which can be used to distinguish pig DNA from that of other animals. When the diagnostic system’s Cas12 endonuclease enzyme encounters the porcine cytochrome b gene, the endonuclease goes into a cutting frenzy, cutting all single-stranded DNA in its neighborhood and activating the test’s fluorescent reporter—a dye molecule tethered by a strand of DNA to a quenching molecule. As Cas12 snips the DNA tethers and releases fluorophores from their quenchers, the solution lights up, indicating how much pork is in the sample by the strength of its fluorescent signal.

Deng’s team found their tool could sense pork DNA down to 1% in mixed meat. DNA from market samples of halal spiced beef and beef lunch meat barely fluoresced above control’s baseline, while DNA from non-halal sausage and dried pork lit up three times as bright.

Compared to qPCR, the CRISPR/Cas test wasn’t as sensitive—qPCR detected as low as 0.1% pork. Yet the CRISPR system requires no temperature cycling and amplification, so it’s dramatically simpler, cheaper, and less likely to become contaminated, Deng says. Because PCR amplifies the very DNA being detected, aerosolized lab contamination from other samples is always a concern, increasing the chance of false positives.

M. A. Motalib Hossain, a researcher who specializes in DNA testing for halal authenticity at the Nanotechnology and Catalysis Research Centre at the University of Malaya, says he appreciates bringing CRISPR into the food authentication area. He notes that the next step would be a CRISPR test that could simultaneously test for pork and other meat counterfeits.

Deng and his team aim to develop the test further so they could take it out of the lab and give it to consumers to do their own testing. The researchers will have to simplify the initial DNA extraction step and find a way to read the fluorescence outside a lab. A cell phone might do, Deng says. “The market for individuals could be huge.”