On September 19, 2025, it was reported that the World Health Organization (WHO) had updated its global situation report on the mpox outbreak. Data showed that as of August 31, 2025, a total of 92 countries had reported 38,671 confirmed cases of mpox and 163 deaths, with the majority concentrated in Africa. A collaborative team led by Professor Dan Du from the School of Medicine, Xiamen University and Professor Youzhi Tang from the College of Veterinary Medicine, South China Agricultural University published a paper in Science Bulletin. Based on the trans-cleavage characteristics of Cas13a and Cas12a enzymes in the CRISPR system, this research established one-pot single-target detection methods by combining them with isothermal amplification RAA technology, respectively. Furthermore, these two systems were integrated to create a dual-target detection system.

The single-target one-pot detection methods using Cas13a and Cas12a demonstrated higher sensitivity and positive detection rates compared to standard quantitative PCR (qPCR), and they do not require complex equipment or trained professional personnel. The dual-target detection system based on Cas13a/Cas12a can be used for multi-target identification of MPXV, reducing the false negative detection rate caused by viral mutations; it can distinguish MPXV subtypes within a single sample, or identify co-infections of MPXV with SARS-CoV-2 or HIV viruses. Testing with clinical simulation samples confirmed the rapid, accurate, reliable, convenient, and efficient characteristics of this method, highlighting its potential for point-of-care testing (POCT) and its contribution to improving disease detection methods and infectious disease management.

The monkeypox virus (MPXV) was first discovered in African non-human primates in the 1950s. The first confirmed human infection with MPXV, causing smallpox-like lesions, was reported in the Republic of Congo in the 1970s. Subsequently, MPXV spread across the African continent, primarily in Central and West Africa. Since cases were reported in Europe in early May 2022, an increasing number of cases have emerged from non-African countries. MPXV is transmitted through contact with infected animals or through direct contact with the body fluids, blood, respiratory droplets, lesions, or contaminated personal objects of natural hosts or patients. Although the symptoms of MPX are generally less severe than those of smallpox, it is still considered a potentially fatal disease. In July 2022, the World Health Organization declared the monkeypox outbreak a Public Health Emergency of International Concern (PHEIC). As of March 2023, over 86,000 confirmed cases and more than 110 deaths have been reported across 110 countries. Facing the threat of the monkeypox epidemic, especially in resource-limited regions, there is an urgent need for rapid and accurate point-of-care diagnostic solutions. Researchers established one-pot single-target detection methods by combining the trans-cleavage characteristics of Cas13a and Cas12a enzymes in the CRISPR system with isothermal amplification RAA technology, respectively, and integrated these two systems to create a dual-target detection system.

1. The detection system can specifically detect the monkeypox virus

Fig. 1. Cas13a-Fluo and Cas12a-Fluo systems specifically detect the monkeypox virus MPXV

The monkeypox virus (MPXV) belongs to the Orthopoxvirus genus, a large family of double-stranded DNA viruses that also includes other viruses such as cowpox virus (CPXV), variola virus (VARV), and ectromelia virus (ECTV). Within the orthopoxvirus genome, the central region is highly conserved, while the terminal regions vary significantly between different viruses. Targeting these variable regions, researchers designed specific crRNAs to recognize the MPXV genome, enabling the specific detection of MPXV among orthopoxviruses using the Cas13a and Cas12a systems, respectively.

2. The detection system can specifically distinguish monkeypox subtypes

Fig. 2. Cas13a-Fluo and Cas12a-Fluo systems specifically distinguish monkeypox virus subtypes

The monkeypox virus (MPXV) is divided into two clades: Central African (MPXV-CA) and West African (MPXV-WA). They have distinct geographical distributions, different clinical manifestations and epidemiological characteristics, and exhibit varying levels of pathogenicity. The Central African clade (MPXV-CA) is associated with a higher fatality rate (approximately 10%), posing a significant public health threat, while the West African clade (MPXV-WA) infection causes milder human disease (with a fatality rate of about 3.6%). Differences in clinical severity may be attributed to genomic variations between the clades. Based on the genomic differences between the two clades, researchers designed different targeting crRNAs. Their corresponding Cas13a and Cas12a detection systems can specifically recognize and distinguish these different viral subtypes, aiding in decision-making for disease prevention and control.

1. Establishment of a one-pot detection method combined with RAA to improve detection sensitivity and reduce aerosol contamination risk

Fig.3. Two types of one-pot detection methods combining isothermal amplification technology RAA with the Cas13a/Cas12a system

To improve the sensitivity of the detection system, researchers combined the isothermal amplification technology RAA with the Cas13a and Cas12a systems. This enhanced the sensitivity of the detection systems while maintaining their specificity. Additionally, point-of-care testing is susceptible to aerosol contamination. Researchers established two types of one-pot reaction detection methods to reduce the risk of aerosol pollution. This one-pot detection method demonstrated higher sensitivity and positive detection rates than standard quantitative PCR (qPCR), requires no complex equipment or trained technical personnel, and is suitable for point-of-care testing in resource-limited areas.

1. Establishment of a dual-target one-pot detection system based on Cas13a/Cas12a

Fig.4. The Cas13a/Cas12a-based dual-target one-pot detection system identifies MPXV, distinguishes MPXV subtypes, and detects MPXV co-infection with SARA-CoV-2/HIV

According to epidemiological data from the World Health Organization (WHO), approximately 51% of monkeypox virus (MPXV) infection cases are also co-infected with the human immunodeficiency virus (HIV). This is likely because both can be transmitted through sexual contact. Lesions caused by MPXV may disrupt tissue mucosal integrity, potentially facilitating HIV transmission. For individuals already infected with HIV and with compromised immune systems, MPXV infection can lead to more severe symptoms and longer disease duration. Research suggests that HIV and MPXV may mutually promote transmission. The Centers for Disease Control and Prevention (CDC) emphasizes that individuals undergoing testing for MPXV infection should also receive HIV testing and prevention services. Therefore, establishing effective early diagnosis and detection is crucial for mitigating the severity of co-infections and controlling the spread of both diseases.

Furthermore, during the COVID-19 pandemic, reported cases of co-infection between monkeypox virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have gradually increased. This may be because both MPXV and SARS-CoV-2 can be transmitted through respiratory droplets; it could also be because SARS-CoV-2 infection compromises the host's immune system, potentially increasing the risk of infection by other viruses. The specific risk of MPXV infection in SARS-CoV-2 positive individuals and the impact of co-infection on symptoms are not yet fully understood. Since MPXV infections primarily occur in resource-limited areas, developing rapid and accurate point-of-care testing methods for detecting co-infections of MPXV with HIV or SARS-CoV-2 will be very valuable for understanding the clinical significance of these co-infections and designing effective treatment and prevention strategies to curb the spread of these diseases.

Researchers integrated the Cas13a and Cas12a systems to establish a one-pot detection system capable of simultaneously detecting dual targets in a single sample. This system can be used for multi-target identification of MPXV, reducing the false negative rate caused by viral mutations; it can distinguish MPXV subtypes within a single sample, improving detection efficiency; or identify co-infections of MPXV with SARS-CoV-2 or HIV viruses, aiding in the effective formulation of corresponding disease prevention and control strategies and treatment measures. Testing with clinical simulation samples confirmed that the method established by the researchers is rapid, accurate, reliable, convenient, and efficient, highlighting its potential for application in point-of-care testing and its contribution to improving disease detection methods and infectious disease management.

Wang X, Rao Q, Lu Z, et al. Rapid and sensitive Cas13a/Cas12a-based one-pot dual-target strategy to detect monkeypox virus and its co-infected viruses. Science Bulletin, 2023.

https://doi.org/10.1016/j.scib.2023.11.023