RESUMEN
On February 19, 2017, China announced that the mutant H7N9 virus appeared in human cases, which showed molecular characteristic of highly pathogenic virus for poultry. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for distinguish between highly pathogenic H7 virus and low pathogenic H7 virus. The sensitivity, specificity, stability and conformance tests were conducted for this method. The data showed that the new method is sensitive. The minimum detection limit for the RNA of highly pathogenic H7 virus is 0.0052fg and the minimum detection limit for the RNA of low pathogenic H7 virus is 0.36fg. The method gave specific results in detecting novel highly pathogenic H7 virus and will play an important role in the rapid identification of novel highly pathogenic H7 virus.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A/aislamiento & purificación , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , China/epidemiología , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Gripe Aviar/diagnóstico , Gripe Aviar/epidemiología , Gripe Aviar/virología , Gripe Humana/diagnóstico , Gripe Humana/epidemiología , Gripe Humana/virología , Técnicas de Diagnóstico Molecular , Aves de Corral/virología , ARN Viral/genética , Sensibilidad y Especificidad , VirulenciaRESUMEN
Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/µl while the LOD for the RT-qPCR was 0.2 PFU/µl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.
Asunto(s)
Virus de la Necrosis Hematopoyética Infecciosa/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Infecciones por Rhabdoviridae/veterinaria , Animales , Cartilla de ADN , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/virología , Virus de la Necrosis Hematopoyética Infecciosa/genética , ARN Viral/aislamiento & purificación , ADN Polimerasa Dirigida por ARN , Infecciones por Rhabdoviridae/diagnóstico , Infecciones por Rhabdoviridae/virología , Sensibilidad y Especificidad , Proteínas del Envoltorio Viral/genética , Carga ViralRESUMEN
H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Enfermedades de las Aves de Corral/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Pollos , Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Aviar/diagnóstico , Enfermedades de las Aves de Corral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Sensibilidad y EspecificidadRESUMEN
This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10â»5(= 280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10â»4(=2800 ELD50). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Animales , Aves , Cartilla de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Gripe Aviar/diagnóstico , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.