New CRISPR-Based Test Achieves Rapid Detection of Tuberculosis

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMAug 1 2024

Despite widespread vaccinations, tuberculosis continues to be a public health concern. Accurate and rapid diagnosis of tuberculosis can significantly improve the treatment and prognosis rates. However, effective diagnosis of tuberculosis is still limited by the lack of sensitive, specific, accurate, and rapid testing methods.

In a recent study published in Microorganisms, a team of researchers developed a recombinase-aided amplification (RAA) assay combined with clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 13a (Cas13a)-based fluorescence assay for the rapid detection of Mycobacterium tuberculosis, the etiological agent of tuberculosis.

​​​​​​​Study: Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System. Image Credit: Sorapop Udomsri/Shutterstock.com

Background

Tuberculosis is a bacterial disease caused by Mycobacterium tuberculosis and is responsible for over 10 million infections and over a million deaths a year.

Accurate diagnostic tests are essential for the rapid diagnosis and timely treatment of tuberculosis and for reducing the prevalence and incidence rates of the disease.

Current diagnostic methods include thoracic imaging, laboratory tests, and analysis of clinical symptoms. Microscopic analysis of sputum smears and culturing M. tuberculosis are the two laboratory techniques predominantly used to detect tuberculosis, primarily due to their ease of use and low costs.

However, inadequate specificity and sensitivity, and the long processing times are major limitations in these methods.

The World Health Organization recommends the Xpert MTB/RIF assay (Xpert) assay, which simultaneously detects the M. tuberculosis complex and rifampicin resistance. However, the high cost and complexity of the system prevent its widespread usage.

The Current Study

In the present study, the researchers presented the RAA-CRISPR-MTB assay that they developed by combining the RAA assay with the CRISPR-Cas13a fluorescence assay to detect M. tuberculosis rapidly.

The CRISPR system evolved as a defense mechanism in bacteria against phages and mobile genetic elements, and it has been combined with various Cas family proteins, such as Cas9 and Cas13, for gene editing and detection of pathogens in vitro based on specific cleavage activity.

The Cas13 structure consists of a recognition lobe domain that binds to the pre-CRISPR ribonucleic acid (pre-crRNA), which then guides the nuclease lobe domain to cleave the target RNA. The CRISPR-Cas13 system was previously used to detect Dengue and Zika viruses with high sensitivity.

Recombinase-aided amplification is an isothermal method of amplifying nucleic acids that does not require precise thermal cycling, unlike those involved in polymerase chain reaction (PCR).

This increases the applications of RAA for molecular diagnostics, especially in laboratory settings lacking complex clinical technology.

For the assay for detecting M. tuberculosis, the researchers developed the RAA-CRISPR-MTB assay by combining RAA's isothermal amplification with the pathogen-detecting abilities of the CRISPR-Cas13a system.

They then evaluated the assay's performance in detecting M. tuberculosis using plasmids, clinical specimens, and M. tuberculosis and non-M. tuberculosis strains. The specificity and sensitivity of the method were compared to those of other diagnostic methods such as Xpert, sputum smears, and M. tuberculosis cultures.

Sputum specimens were obtained from active tuberculosis patients and subjected to rapid deoxyribonucleic acid (DNA) extraction. A plasmid template for positive control was also prepared using a partial IS6110 gene sequence from M. tuberculosis and the H37Rv strain.

After preparing the crRNA, the RAA-CRISPR-MTB was verified by amplifying the sample DNA templates through RAA and detecting the IS6110 gene sequence through the CRISPR-Cas13a assay.

Major Findings

The study found that the RAA-CRISPR-MTB assay could detect M. tuberculosis at one copy per microliter level when the plasmid carrying the partial IS6110 gene sequence was used as the template.

When the H37Rv strain was used as the template, the RAA-CRISPR-MTB assay detected M. tuberculosis at ten copies per microliter. Furthermore, no cross-detection involving non-tuberculosis Mycobacterium species was observed with either of the templates.

The diagnostic sensitivity of the RAA-CRISPR-MTB assay was 69% when clinical specimens were used for M. tuberculosis diagnosis, which was higher than the sensitivity of the Xpert system (60%). However, the RAA-CRISPR-MTB assay showed 100% specificity.

The use of the IS6110 gene insertion sequence enhanced the specificity and sensitivity of the assay since the sequence is present only in the genomes of members of the M. tuberculosis complex.

Furthermore, the sequence is present in multiple copies in the genome, which provides better detection specificity than assays that use single-copy genes.

While the RAA-CRISPR-MTB assay eliminates the need for precise thermal control equipment, the dependence on trained professionals and specialized equipment continues to present challenges in the application of this assay in low-resource settings.

Therefore, the researchers intend to focus their future research on developing customized lateral flow strips using the RAA-CRISPR-MTB assay that can be used in non-specialized clinical settings.

Conclusions

Overall, the study showed that the RAA-CRISPR-MTB assay developed using recombinase-aided amplification and the CRISPR-Cas13a system effectively detected M. tuberculosis with high specificity and sensitivity compared to existing methods.

This assay also has the potential to be adapted for use in non-clinical settings, contributing to more effective detection and treatment of tuberculosis.