Using two pairs of primer based on conserved sequences of the NP gene of influenza A virus, a RT-nested PCR assay was developed to detect Swine influenza virus in clinical samples. No cross reactions with three other ...Using two pairs of primer based on conserved sequences of the NP gene of influenza A virus, a RT-nested PCR assay was developed to detect Swine influenza virus in clinical samples. No cross reactions with three other swine pathogens of respiratory disease such as Porcine respiratory and reproductive syndrome virus, Porcine circovirus type 2 and Mycoplasma suipneumoniae. With agarose electrophoresis detection, the RT-nested PCR was 100 times more sensitive compared to that of non-nested RT-PCR. 35 clinical samples including 4 blood samples and 31 lung tissues from Southern China were tested by RT-nested PCR, and 28 (80%) out of 35 sample were positive. By propagated in 9-to 10-day-old SPF embryonated chicken eggs, 23 strains of Swine influenza virus were isolated from 28 positive samples. The result of RT-nested PCR was 82% in agreement with that of virus isolation, and the RT-nested PCR were more sensitive than the virus isolation. No Swine influenza virus was isolated from the all RT-nested PCR negative samples. On the basis of characterization of HA gene sequences, 5 out of 23 strains had H3 and H9 subtypes. It suggested that the RT-nested PCR assay was rapid,specific and sensitive, and it was is advantageous to detect influenza virus in latent infections.展开更多
A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Speci...A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.展开更多
文摘Using two pairs of primer based on conserved sequences of the NP gene of influenza A virus, a RT-nested PCR assay was developed to detect Swine influenza virus in clinical samples. No cross reactions with three other swine pathogens of respiratory disease such as Porcine respiratory and reproductive syndrome virus, Porcine circovirus type 2 and Mycoplasma suipneumoniae. With agarose electrophoresis detection, the RT-nested PCR was 100 times more sensitive compared to that of non-nested RT-PCR. 35 clinical samples including 4 blood samples and 31 lung tissues from Southern China were tested by RT-nested PCR, and 28 (80%) out of 35 sample were positive. By propagated in 9-to 10-day-old SPF embryonated chicken eggs, 23 strains of Swine influenza virus were isolated from 28 positive samples. The result of RT-nested PCR was 82% in agreement with that of virus isolation, and the RT-nested PCR were more sensitive than the virus isolation. No Swine influenza virus was isolated from the all RT-nested PCR negative samples. On the basis of characterization of HA gene sequences, 5 out of 23 strains had H3 and H9 subtypes. It suggested that the RT-nested PCR assay was rapid,specific and sensitive, and it was is advantageous to detect influenza virus in latent infections.
基金supported by a grant from the Out-standing Person Innovation Foundation of Henan,China(0621002100)
文摘A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.
