Development and validation of real-time recombinase polymerase amplification-based assays for detecting HPV16 and HPV18 DNA.

IMPORTANCE: HPV DNA screening is an effective approach for the prevention of cervical cancer. The novel real-time recombinase polymerase amplification-based HPV detection systems we developed constitute an improvement over the HPV detection methods currently used in clinical practice and should help to extend cervical cancer screening in the future, particularly in point-of-care test settings.

Rapid Detection of the Varicella-Zoster Virus Using a Recombinase-Aided Amplification-Lateral Flow System.

Varicella-zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). VZV infections are ubiquitous and highly contagious, and diagnosis is mostly based on the assessment of signs and symptoms. However, monkeypox, an emerging infectious disease caused by the monkeypox virus (MPXV), has clinical manifestations that are similar to those of VZV infections. With the recent monkeypox outbreak in non-endemic regions, VZV infections are likely to be misdiagnosed in the absence of laboratory testing. Considering the lack of accessible diagnostic tests that discriminate VZV from MPXV or other poxviruses, a handy and affordable detection system for VZV is crucial for rapid differential diagnosis. Here, we developed a new detection method for VZV using recombinase-aided amplification technology, combined with the lateral flow system (RAA-LF). Given the prevalence of VZV worldwide, this method can be applied not only to distinguish VZV from other viruses causing rash, but also to foster early detection, contributing substantially to disease control.

Development and Characterization of Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of Monkeypox Virus.

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), in which outbreaks mainly occurred in West and Central Africa, with only sporadic spillovers to countries outside Africa due to international travel or close contact with wildlife. During May 2022, multiple countries in Europe, North and South America, Australia, Asia, and Africa reported near-simultaneous outbreaks of MPXV, the first time that patient clusters were reported over such a large geographical area. Cases have no known epidemiological links to MPXV-endemic countries in West or Central Africa. Real-time PCR is currently the gold standard for MPXV diagnostics, but it requires trained laboratory personnel and specialized equipment, and results can only be obtained after several hours. A rapid and simple-to-operate point-of-care diagnostic test for MPXV is crucial for limiting its spread and controlling outbreaks. Here, three recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of MPXV isolates were developed. These three assays target the MPXV G2R gene, and the limit of detection for these systems is approximately 100 copies of DNA per reaction. The assays were found to be specific and non-cross reactive against other pox viruses, such as vaccinia virus, and the results can be visualized within 20-30 min. The assays were validated with DNA extracted from 19 clinical samples from suspected or confirmed MPXV patients from Central Africa, and found to be consistent with findings from traditional qPCR. These results provide a solid platform for the early diagnosis of potential MPXV cases, and will help with the control and prevention of current and future outbreaks.

Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections.

A recent outbreak of monkeypox virus (mpox) has prompted researchers to explore diagnostics as a means of impeding transmission and further spread. Rapid, sensitive, and specific methods are crucial for accurately diagnosing mpox infections. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of mpox. The primer sets were designed to target regions in and around the N4R gene, and results showed a detection limit of 2 × 100 DNA copies, which is comparable to the gold-standard qPCR method currently used to detect mpox. Particularly, the assay provides results visible to the naked eye within 30 min. This test specifically detects mpox DNA with no cross-reactivity to related DNA viruses including Varicella Zoster Virus (VZV), Hepatitis B virus (HBV), Vaccinia virus (VACV), Herpes simplex virus-1 (HSV-1), Herpes simplex virus-2 (HSV-2), Human papillomavirus-16 (HPV-16) and Human papillomavirus-18 (HPV-18). Furthermore, the LAMP assay has been evaluated using clinical samples from laboratory-confirmed mpox patients and found to be consistent with the qPCR results. Our results show that this single-tube LAMP method can contribute to diagnosis of suspected mpox infections in the field and clinic, especially in regions with limited laboratory resources.

Chemical composition, antioxidant, antibacterial and cytotoxic activities of essential oil of Leontopodium leontopodioides (Willd.) Beauverd.

The essential oil (EO) from the aerial parts of Leontopodium leontopodioides (Willd.) Beauverd was obtained by hydrodistillation and analysed by GC-FID and GC-MS. Sixty-five compounds were identified which represent 96.2% of the total composition of the EO. The major components of the EO were palmitic acid (11.6%), n-pentadecanal (5.7%), linalool (3.8%), ß-ionone (3.3%), hexahydrofarnesyl acetone (3.2%), bisabolone (3.2%) and ß-caryophyllene (3.2%). The EO exhibited an excellent antibacterial activity against Staphylococcus aureus and Bacillus subtilis according to the MIC values tested by micro-dilution method. It also exhibited a significant cytotoxicity against HepG2 and MCF-7 cell lines with the IC50 values of 67.44 and 70.49 µg/mL according to the MTT assay. However, the antioxidant activity test revealed that the EO exhibited a weak DPPH radical-scavenging activity. In conclusion, the EO of L. leontopodioides could be regarded as a bioactive natural product and deserves further study for its potential therapeutic effects.