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Zhang Y.,CAS National Center for Nanoscience and Technology | Zhang Y.,Peking University | Zhang L.,CAS National Center for Nanoscience and Technology | Sun J.,CAS National Center for Nanoscience and Technology | And 6 more authors.
Analytical Chemistry | Year: 2014

This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. © 2014 American Chemical Society.


Song C.,Ocean University of China | Song C.,China Animal Health and Epidemiology Center | Zhu C.,Harbin Veterinary Research Institute of CAAS | Zhang C.,Harbin Veterinary Research Institute of CAAS | Cui S.,Harbin Veterinary Research Institute of CAAS
Virology Journal | Year: 2010

A TaqMan-based real-time polymerase chain reaction (PCR) assay was devised for the detection of porcine parvovirus (PPV). Two primers and a TaqMan probe for the non-structural protein NS1 gene were designed. The detection limit was 1 × 102 DNA copies/L, and the assay was linear in the range of 1 × 102 to 1 × 109 copies/L. There was no cross-reaction with porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The assay was specific and reproducible. In 41 clinical samples, PPV was detected in 32 samples with the real-time PCR assay and in only 11 samples with a conventional PCR assay. The real-time assay using the TaqMan-system can therefore be practically used for studying the epidemiology and management of PPV. © 2010 Song et al.


Rui-Hua Z.,Hebei North University | Hong-Yu C.,Hebei North University | Ming-Ju X.,Hebei North University | Kai L.,Hebei North University | And 5 more authors.
Acta Virologica | Year: 2011

The H9N2 subtype influenza virus (IV) is a remarkable member of the influenza A viruses because it can infect not only chickens, ducks and pigs, but also humans. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. To further understand the genetic characteristics and evolution, we investigated the source and molecular characteristics of the H9N2 subtype swine influenza virus (SIV), and observed its pathogenicity in BALB/c mice. The BALB/c mice were inoculated intranasally with 100 median mouse infectious dose of A/swine/HeBei/012/2008/(H9N2) viruses to observe the pathogenicity. The HA, NP, NA and M gene were cloned, sequenced and phylogenetically analyzed with related sequences available in GenBank. The infected mice presented with inactivity, weight loss and laboured respiration, while the pathological changes were characterized by diffuse alveolar damage in the lung. The nucleotide and deduced amino acid sequence of HA, NP, NA and M gene was similar with that of A/chicken/Hebei/4/2008(H9N2). The HA protein contained 6 glycosylation sites and the motif of HA cleavage site was PARSSR GLF, which is characteristic of low pathogenic IV. In the HA, NP, M and NA gene phylogenetic trees, the isolate clustered with A/chicken/Hebei/4/2008(H9N2). The isolate possibly came from A/chicken/Hebei/4/2008(H9N2) and was partially varied during its cross-species spread.


Zhou H.,Northwest University, China | Yao G.,Harbin Veterinary Research Institute of CAAS | Cui S.,Harbin Veterinary Research Institute of CAAS
Virology Journal | Year: 2010

The porcine parvovirus (PPV) VP2 protein was expressed in an insect-baculovirus cell system and was purified using Ni-NTA affinity column chromatography. The recombinant 6-His-tagged VP2 protein with molecular mass (Mr) of about 64 kDa was detected by anti-his antibody and anti-PPV serum. Electron microscopy showed that the purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The expressed VP2 was antigenically similar to the native capsid protein according to HA and a Western blotting assay performed with polyclonal antibodies collected from an outbreak of PPV in one farm. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of PPV or in the vaccination against PPV in the future. © 2010 Zhou et al; licensee BioMed Central Ltd.


Chen C.,Harbin Veterinary Research Institute of CAAS | Cui S.,Harbin Veterinary Research Institute of CAAS | Zhang C.,Harbin Veterinary Research Institute of CAAS | Li J.,Shandong Academy of Agricultural Sciences | Wang J.,Shandong Academy of Agricultural Sciences
Virus Genes | Year: 2010

To establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV), four primers specific to six regions of the N gene were designed. After amplification in an isothermal water bath for 1 h, samples containing PRRSV generated the expected ladder-like products while porcine parvovirus, porcine circovirus, classic swine fever virus, pseudorabies virus, and swine testis cells generated no product. The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Because it is specific and simple, the RT-LAMP assay will be useful for the diagnosis of PRRSV infection. © 2009 Springer Science+Business Media, LLC.


PubMed | Harbin Veterinary Research Institute of CAAS
Type: Journal Article | Journal: Veterinary microbiology | Year: 2012

The preparation of high-yield influenza H5N1 vaccine strains is challenging for researchers and manufacturers. Here, we used reverse genetics to generate a high-yield avian influenza vaccine strain based on a novel avian influenza virus. A high-yield attenuated recombinant H5N3 virus (rH5N3-DL) was prepared from the HA gene of A/Goose/Anhui/08 (H5N1), modified by deletion of the multiple basic amino acids at the cleavage site, the NA gene from A/Duck/Germany/1215/73 (H2N3), and the six internal genes from the high-yield A/Goose/Dalian/3/01 (H9N2) virus. rH5N3-DL grew to high HA titers (1:2048) in eggs, eight times those of the parental H5N1 virus, and four times higher than that of rH5N3-PR8 (six internal genes from the high-yield PR8). Infection tests demonstrated that rH5N3-DL was avirulent in chickens, chicken embryos, and mice. rH5N3-DL-vaccinated chickens were fully protected against the morbidity and mortality of a lethal challenge with homologous A/Goose/Anhui/08, but only 80% of chickens were protected after challenge with heterologous A/Goose/Guangdong/1/96. The N3 neuraminidase marker distinguishes rH5N3-DL-vaccinated from H5N1-infected animals. rH5N3-DL is thus a promising vaccine candidate to combat highly pathogenic avian influenza virus infections. The A/Goose/Dalian/3/01 virus could be a promising candidate as providing internal genes donors with high-yield properties in reverse-genetics system and might be applicable for future avian influenza vaccine development.


PubMed | Harbin Veterinary Research Institute of CAAS
Type: Journal Article | Journal: Journal of virological methods | Year: 2010

A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). One group of primers was designed to detect wild-type strains (i.e., strains with the gE gene) and the other group of primers was designed to detect both PRV gE-vaccine and wild-type strains (i.e., strains with the gG gene and with or without the gE gene). After amplification by Bst enzyme at a constant temperature of 65 degrees C, a laddering of bright products was visible following electrophoresis on a 2% agarose gel. LAMP was 100-1000-fold more sensitive than the standard PCR. The assay was specific in that it did not amplify other porcine viruses including porcine parvovirus, porcine circovirus type 1, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus, swine transmissible gastroenteritis coronavirus, and porcine epidemic diarrhea virus. Because of its sensitivity, specificity, and simplicity, the LAMP assay could be a useful method for early and rapid differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus.


PubMed | Harbin Veterinary Research Institute of CAAS
Type: Journal Article | Journal: Sheng wu gong cheng xue bao = Chinese journal of biotechnology | Year: 2010

According to 45 hemagglutinin (HA) gene sequences of H7 subtype of avian influenza virus (AIV), a pair of specific oligonucleotide primers was designed. We developed one step RT-PCR for detecting AIV subtype H7. Sensitivity to detection of allantoic fluid by one step RT-PCR reached 10(5.5) EID50/mL and detection of swab samples reached 10(3) EID50/mL. We simultaneity detected the tissue and swab samples infected with H7 subtypes AIV by one step RT-PCR and virus isolation method. The results showed that the sensitivity of the assay gave an excellent correlation with the conventional virus isolation method. H1-H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR. The results showed the assays were high specific, without cross-reaction with other subtypes or other avian diseases. Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of AIV subtype H7.


PubMed | Harbin Veterinary Research Institute of CAAS
Type: Journal Article | Journal: Virus genes | Year: 2010

To establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV), four primers specific to six regions of the N gene were designed. After amplification in an isothermal water bath for 1 h, samples containing PRRSV generated the expected ladder-like products while porcine parvovirus, porcine circovirus, classic swine fever virus, pseudorabies virus, and swine testis cells generated no product. The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Because it is specific and simple, the RT-LAMP assay will be useful for the diagnosis of PRRSV infection.


PubMed | Harbin Veterinary Research Institute of CAAS
Type: Journal Article | Journal: Veterinary immunology and immunopathology | Year: 2012

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.

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