RESUMEN
Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium hepatitis syndrome in chickens, which causes severe economic impact to the poultry industry. A simple, swift and reliable detection is crucial for timely identification of FAdV-4 infection, promoting effective viral prevention and control measures. Herein, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system detection platform based on loop-mediated isothermal amplification (LAMP) was studied. The CRISPR RNA (crRNA) and LAMP primers were designed and screened based on the highly conserved region of the FAdV-4 hexon gene. The parameters were then optimized individually to achieve the ideal reaction performance. The platform could lead visual detection of FAdV-4 to achieve as low as 1 copy in less than 40 min without the need for specialized instrumentation or complex equipment. Moreover, it was greatly specific, and did not cross-react with other common avian viruses. Following the validation of 30 clinical samples of suspected FAdV-4 infection, the results LAMP-CRISPR/Cas12a method generated showed fully concordance with which of the gold standard quantitative real-time PCR. To summarize, this study presented a novel, swift, expedient and inexpensive detection platform for FAdV-4, which is beneficial to viral inchoate diagnosis and point-of-care testing.
RESUMEN
Since 2015, an outbreak of an infectious disease in broilers caused by fowl adenovirus serotype 4 (FAdV-4) has occurred in China, resulting in substantial economic losses. Rapid, accurate, and specific detection are significant in the prevention and control of FAdV-4. In this study, an FAdV-4 detection method combining loop-mediated isothermal amplification (LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established. Specific primers, guide DNAs (gDNAs), and molecular beacons were designed to target a conserved region of the FAdV-4 hexon gene. After optimizing the reaction conditions, the minimum detection of this assay could reach 5 copies. It only amplified FAdV-4, and there was no cross-reactivity with other pathogens. The assay took about only 50 min, and the results could be visualized with the naked eye under ultraviolet or blue light, getting rid of specialized instruments. This novel LAMP-PfAgo assay was validated by using 20 clinical samples and the results were identical to gold-standard real-time polymerase chain reaction method. In summary, the LAMP-PfAgo assay established in the paper provides a rapid, reliable, convenient, ultra-sensitive and highly specific tool for the on-site detection and clinical diagnosis of FAdV-4.
Asunto(s)
Infecciones por Adenoviridae , Aviadenovirus , Pollos , Técnicas de Amplificación de Ácido Nucleico , Enfermedades de las Aves de Corral , Pyrococcus furiosus , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones por Adenoviridae/veterinaria , Infecciones por Adenoviridae/virología , Infecciones por Adenoviridae/diagnóstico , Animales , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/diagnóstico , Pyrococcus furiosus/genética , Aviadenovirus/genética , Aviadenovirus/aislamiento & purificación , Aviadenovirus/clasificación , Sensibilidad y Especificidad , Serogrupo , Proteínas Argonautas/genética , Técnicas de Diagnóstico Molecular/veterinaria , Técnicas de Diagnóstico Molecular/métodosRESUMEN
A recent outbreak of porcine circovirus-like virus (PCLV), a virus that may be associated with porcine diarrhea, has been reported in swine herds in China. The virus is spreading rapidly, causing huge economic losses to the swine farming industry. To achieve the rapid, inexpensive, and sensitive detection of PCLV, we combined loop-mediated isothermal amplification (LAMP) and the CRISPR/Cas12a system, whose fluorescence intensity readout can detect PCLV ORF4 gene levels as low as 10 copies. To overcome the need for sophisticated equipment, lateral flow strip reading technology was introduced for the first time in a LAMP-Cas12a-based system to detect PCLV. The lateral flow strip (LFS) results were readout by the naked eye, and the method was highly sensitive with a detection limit of 10 copies, with a detection time of about 60 min. In addition, the method is highly specific and has no cross-reactivity with other related viruses. In conclusion, LAMP-CRISPR/Cas12a-based assays have the advantages of rapidity, accuracy, portability, low cost, and visualization of the results. They therefore have great potential, especially for areas where specialized equipment is lacking, and can expect to be an ideal method for early diagnosis and on-site detection of PCLV.
