>Highlights The RT-RAA-VF assay developed for the NiV P gene can perform rapid detection of NiV within 20 min at 42°C with high specificity.This assay is capable of attaining sensitivity to a single copy of NiV RNA transcripts.This assay effectively avoids false positives caused by aerosol contamination with a sealed disposable nucleic acid visualization test paper device.

Nipah virus (NiV) is an emerging bat-borne zoonotic virus that can be transmitted to humans and other animals through infected bats or contaminated foods. The disease is highly lethal in humans (40%-75%) and has the potential for human-to-human transmission. Currently, there are no approved treatments or vaccines for NiV infection in humans or animals. Consequently, there is a pressing need for a highly sensitive, precise, and visually detectable assay to enable early intervention and mitigate the transmission of NiV infection. Here, we report a single-copy sensitive, field-deployable, one-pot visual reverse transcription-recombinase polymerase amplification (RT-RPA)-clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associate system (Cas)12 for the detection of NiV. The assay works by targeting the N gene of NiV, and the results are directly visible to the naked eye. The assay has demonstrated the ability to detect as few as 5.5 copies/μl of positive plasmids or 5.5 × 101 copies/μl of RNA transcripts when reacted at constant temperature for 40 min. It showed high specificity for NiV and had no cross-reaction with other pathogens, including rabies virus (RABV), Japanese encephalitis virus (JEV), herpes simplex virus type 1 (HSV-1), Hendra virus (HeV), and Streptococcus suis (S. suis), that can cause clinical symptoms similar to those of NiV infection. Moreover, this assay had a 100% coincidence rate with the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method recommended by the World Organization for Animal Health (WOAH) for the detection of simulated clinical samples, indicating that it has great potential as an ultrasensitive, simple, and portable novel assay for the onsite diagnosis of NiV infection.

Crimean-Congo hemorrhagic fever（CCHF） is a zoonotic disease caused by the CCHF virus（CCHFV）, which is primarily transmitted by ticks（Lorenzo Juanes et al. 2023）. It is an emerging disease that occurs sporadically in Africa, Asia, and Europe, with a high morbidity and mortality rate, as high as 30% in humans（Ceylan et al. 2013）. CCHFV, belonging to genus Nairovirus,

•A MPXV visual assay panel is a rapid and reliable tool to differentiate the clades I and II within 25 min.•This panel combines RAA and immunochromatography, and detects as low as 1 copy/μL recombinant plasmid.•Visual assay panel shows no cross-reactivity with orthopoxviruses and herpesvirus that infect humans, such as vaccinia virus.

Monkey B virus (BV) infection in humans and other macaque species has a mortality rate of approximately 80%. Because BV infects humans through bites, scratches, and other injuries inflicted by macaques, the simple and rapid diagnosis of BV in field laboratories is of great importance to protect veterinarians, laboratory researchers, and support personnels from the threat of infection. Two recombinase polymerase amplification (RPA) assays with a closed vertical flow (VF) visualization strip (RPA-VF-UL27 and RPA-VF-US6) were developed that target two conserved genes combined with a one-off, closed visualization strip device. We compared the sensitivities and specificities of the two assays after optimization of the reaction conditions. The performance of RPA-VF-US6 at room temperature was determined to evaluate its potential in point-of-care (POC) testing. RPA-VF-US6 specifically detected the positive plasmid control (rather than nucleic acids of herpesviruses) with a detection limit of 28 copies, while RPA-VF-UL27 had cross-reactivity with HSV-1, but even 3.4 copies of plasmid standards were readout by this assay. Moreover, RPA-VF-US6 had excellent performance at room temperature (the detection limit was 2,800 plasmid copies), indicating the potential of RPA-VF-US6 in POC testing. We developed two RPA assays for BV visualization diagnosis. RPA-VF-US6 is a simple, rapid, and specific detection method for BV. The entire reaction can be performed at a constant temperature within 30 min, suggesting the potential of RPA-VF-US6 for POC testing in field laboratories without sophisticated instruments.

IBRV causes different degrees of clinical symptoms in cattle and poses a great threat to the cattle industry. The infection is persistent and latent, and the elimination of IBRV in infected herds is difficult.

本试验运用多重PCR检测技术检测奶牛乳房炎乳汁中的金黄色葡萄球菌(Staphylococcus aureus)、无乳链球菌(Streptococcus agalactiae)和绿脓杆菌(Pseudfomonas aeruginosa)3种病原菌,确认该方法在乳汁病原菌检测中的适用性和有效性,并探讨病原菌的感染情况.选取四川奶牛场送检的191份奶样(经过加州乳房炎检测法检测,其中16份为正常奶样,64份为隐性乳房炎奶样,111份为临床型乳房炎奶样),分别进行多重PCR法和传统细菌学培养法检测,并对照和分析2种方法的差异.结果显示,多重PCR检测法检出135株致病菌,检出率为70.7％;传统细菌学培养法检出99株致病菌,检出率为51.8％;通过卡方分析证明2种检测方法的检出率差异显著(P＜0.05).鉴于多重PCR法表现出较强的特异性、灵敏度,且操作方便,非常适用于3种混合感染病原菌在奶牛临床型乳房炎、隐性乳房炎奶样的检测和流行病学调查.