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Three isolates of H9N2 subtype of influenza virus have been isolated from chickens in Guangxi province. In this study, eight full-length genes of each of the H9N2 isolates, A/Chicken/Guangxi/1/00, A/Chicken/Guangxi/14/00, and A/Chicken/Guangxi/17/00, were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes of each isolate with those of the available H9N2 strains at GenBank. Results showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that Guangxi isolates originated from the same source. However, the eight genes of these three isolates from Guangxi were not in the same sublineages in the phylogenic trees, which suggest that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA encoding amino acids T, E, I were absent between nucleotide 205 and 214 in the open reading frame of NA genes in the Guangxi isolates. AIV strains that infect human have in their HA proteins, Leucine at position 226. The analysis of deduced amino acid sequence of HA proteins of C/GX/1/00, C/GX/14/00, and C/GX/17/00 showed that the position 226 of these isolates was glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from a pigeon in Guangxi, southern China, in 2014. The eight RNA segment genes shared a high nucleotide identity (97 to 99%) with H6N6 subtypes of AIV isolated from ducks in the regions around Guangxi Province. The finding of this study will help us understand the ecology and molecular characteristics of H6 avian influenza virus in wild birds in southern China.

Objectives In order to develop a multiplex RT‐PCR assay using the GeXP analyser for the simultaneous detection of four different NA serotypes of H5‐subtype AIVs, effective to control and reduce H5 subtype of avian influenza outbreak. Design Six pairs of primers were designed using conserved and specific sequences of the AIV subtypes H5, N1, N2, N6 and N8 in GenBank. Each gene‐specific primer was fused at the 5′ end to a universal sequence to generate six pairs of chimeric primers, and one pair of universal primers was used for RT‐PCR, and PCR product separation and detection were performed by capillary electrophoresis using the GenomeLab GeXP genetic analysis system. Setting Single and mixed avian pathogen cDNA/DNA templates were employed to evaluate the specificity of a multiplex assay with a GeXP analyser. Corresponding specific DNA products were amplified for each gene, revealing amplification peaks for M, H5, N1, N2, N6 and N8 genes from four different NA subtypes of influenza A H5 virus. Sample A total of 180 cloacal swabs were collected from poultry at live bird markets. Main outcome measures The multiplex PCR assay demonstrated excellent specificity, with each pair of specific primers generating only products corresponding to the target genes and without cross‐amplification with other NA‐subtype influenza viruses or other avian pathogens. Using various premixed ssRNAs containing known AIV target genes, the detection limit for the multiplex assay was determined to be 102 copies/μl. The GeXP assay was further evaluated using 180 clinical specimens and compared with RRT‐PCR (real‐time reverse transcriptase PCR) and virus isolation. Conclusions This GeXP analyser‐based multiplex assay for four different NA subtypes of H5 HPAI viruses is both highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples.

A field strain of H3N6 avian influenza virus (AIV), A/duck/Guangxi/175D12/2014(H3N6), was isolated from a native duck in Guangxi Province, southern China, in 2014. All of the eight AIV gene segments were sequenced, and sequence results revealed that there were 11 amino acid deletions at the NA stalk region. The NA, PB2, and NP genes showed highest homology to H5N6 AIV, and the PA gene showed highest homology to H7N2 AIV. Phylogenetic analysis indicated that the eight AIV gene segments belonged to the Eurasian lineage. These findings provide scientific evidence of possible or potential mutations of H3N6 AIV circulating in waterfowl in southern China.

Background Duck viral pathogens primarily include the avian influenza virus (AIV) subtypes H5, H7, and H9; duck hepatitis virus (DHV); duck tembusu virus (DTMUV); egg drop syndrome virus (EDSV); duck enteritis virus (DEV); Newcastle disease virus (NDV); duck circovirus (DuCV); muscovy duck reovirus (MDRV); and muscovy duck parvovirus (MDPV). These pathogens cause great economic losses to China’s duck breeding industry. Result A rapid, specific, sensitive and high-throughput GeXP-based multiplex PCR assay consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed and optimized to simultaneously detect these eleven viral pathogens. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Corresponding specific DNA products were amplified from each pathogen. Other pathogens, including duck Escherichia coli, duck Salmonella, duck Staphylococcus aureus, Pasteurella multocida, infectious bronchitis virus, and Mycoplasma gallisepticum, did not result in amplification products. The detection limit of GeXP was 103copies when all twelve pre-mixed plasmids containing the target genes of eleven types of duck viruses were present. To further evaluate the reliability of GeXP, 150 clinical field samples were evaluated. Comparison with the results of conventional PCR methods for the field samples, the GeXP-multiplex PCR method was more sensitive and accurate. Conclusion This GeXP-based multiplex PCR method can be utilized for the rapid differential diagnosis of clinical samples as an effective tool to prevent and control duck viruses with similar clinical symptoms.

The objective of this study was to obtain the complete mitochondrial DNA sequence of chukar partridge, and to provide reference data for protection and utilization of these resources of chukar partridge. The complete mitochondrial genome sequence of the China chukar partridge was measured by PCR-based methods and analysed in detail. Our research findings reveal that the entire mitochondrial genome of the chukar partridge is a circular molecule consisting of 16,688 bp (GenBank accession number: KY829450). The contents of A, T, C, and G in the mitochondrial genome were found to be 30.44%, 24.43%, 31.57%, and 13.56%, respectively. The complete mitochondrial genome of the chukar partridge has a typical structure, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one control region (D-loop region). This complete mitochondrial genome sequence provides essential information in understanding phylogenetic relationships among Galliformes mitochondrial genomes.

The H3 subtype of avian influenza virus can provide genes for human influenza virus through gene reassortment, which has raised great concerns about its potential threat to human health. An H3N6 subtype of avian influenza virus was isolated from Guangxi Province, China, in 2009. All eight gene segments of the strain were sequenced. The sequence analysis indicated that this H3N6 virus was a nature reassortant virus. The genome sequences now can be used to understand the epidemiological and molecular characteristics of the H3N6 influenza virus in southern China.

A duck tembusu virus (DTMUV) was isolated from the brain of a Cherry Valley duckling that showed neurological signs by using a specific-pathogen-free chicken embryo. The isolate was named GX2013G (GenBank accession no. KM275941). The strain GX2013G was identified with reverse transcription-PCR (RT-PCR), and the amplicon was sequenced. The genome that was obtained is 10,990 nucleotides in length and contains a single open reading frame encoding a putative polyprotein of 3,425 amino acids. This study will advance the understanding of the epidemiology and molecular characteristics of tembusu virus (TMUV) in Guangxi and further studies of the mechanisms of virus replication and pathogenesis.

The complete mitochondrial genome sequence of the Cenxi classical three-buff chicken was measured by PCR-based methods and analyzed in detail. Our research findings reveal that the entire mitochondrial genome of the Cenxi classical three-buff chicken is a circular molecule consisting of 16,786 bp (GenBank accession number: KM433666). The contents of A, T, C, and G in the mitochondrial genome were found to be 30.27%, 23.75%, 32.49% and 13.49%, respectively. The complete mitochondrial genome of the Cenxi classical three-buff chicken contains a typical structure, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region (D-loop region). This complete mitochondrial genome sequence provides essential information in understanding phylogenetic relationships among Gallus gallus domesticus mitochondrial genomes.

We report here two complete genome sequences of porcine circovirus 2. The complete genomes of BH5 and BH6 were amplified and analyzed. Sequence analysis demonstrated that strains BH5 and BH6 belonged to PCV2d and PCV2b, respectively. Knowledge regarding the complete genome sequences of strains BH5 and BH6 will be useful for epidemiological surveillance.

The complete mitochondrial genome sequence of the Rongshui Xiang duck was measured by PCR-based methods. Our research findings reveal that the entire mitochondrial genome of the Rongshui Xiang duck is a circular molecule consisting of 16,605 bp (GenBank accession number: KJ833587). The contents of A, T, C, and G in the mitochondrial genome were found to be 29.20%, 22.20%, 32.82% and 15.79%, respectively, similar to the majority of avian species. The complete mitochondrial genome of the Rongshui Xiang duck contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region. The characteristics of the mitochondrial genome were analyzed in detail. Our complete mitochondrial genome sequence should provide essential information for understanding phylogenetic relationships among duck mitochondrial genomes.

The complete mitochondrial genome sequence of the Xilin small partridge duck was measured by PCR-based methods. Our research findings revealed that the entire mitochondrial genome of the Xilin small partridge duck was 16,604 bp (GenBank accession number: KJ833586). The contents of A, T, C, and G in the mitochondrial genome were 29.20%, 22.19%, 32.82% and 15.79%, respectively, which were similar to the majority of most avian species. The complete mitochondrial genome of the Xilin small partridge duck contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region. Components of the Xilin small partridge duck’s mitochondrial genome were similar to those of other Anatidae in gene arrangement and composition. The complete mitochondrial genome of the Xilin small partridge duck should provide essential information for understanding phylogenetic relationships of duck mitochondrial genome.

A H3N8 subtype of avian influenza virus, A/goose/Guangxi/020G/2009(H3N8) (GX020G), was isolated from the Guangxi Province of China in 2009. All eight gene segments of the GX020G strain were sequenced. Sequence analysis indicated that this H3N8 virus is a novel reassortant strain. The genome sequences provide useful information for understanding the epidemiology and molecular characteristics of the H3N8 subtype of influenza virus in southern China.

We report here the complete genome sequence of a duck Tembusu virus (DTMUV) strain, GX2013H, isolated from a duck from Cheery Valley in the Guangxi Province of southern China in 2013. We obtained the strain GX2013H from a Cheery Valley duck with severely decreased egg production and neurological signs. The genome of GX2013H is 10,990 nucleotides (nt) in length and contains a single open reading frame encoding a putative polyprotein of 3,425 amino acids (aa). A comparison of the complete sequence and the deduced amino acid sequence of GX2013H with published sequences of 15 other chicken anemia viruses from China showed that the homologies of the nucleotides are approximately 96.5% to 97.5% and the homologies of the deduced amino acid sequences are approximately 98.9% to 99.3%. This report will help to understand the epidemiology and molecular characteristics of TMUV in Guangxi.

We report here the full-length RNA genomic sequence of the bovine viral diarrhea virus (BVDV) strain GX4, isolated from a cow in southern China. Studies indicate that BVDV GX4 belongs to the BVDV-1b subtype. This report will help in understanding the epidemiology and molecular characteristics of BVDV in southern China cattle.

We report here the complete genome sequence of a triple-reassortant H1N1 swine influenza virus strain, A/swine/Guangxi/BB1/2013 (H1N1) (GXBB1), isolated from a swine in the Guangxi Province of southern China in 2013. We obtained the complete genome sequence of the GXBB1 virus. Sequence analysis demonstrated that this H1N1 virus was a triple-reassortant swine influenza virus (SIV) whose genes originated from avian, human, and swine, respectively. Knowledge regarding the complete genome sequence of the GXBB1 virus will be useful for epidemiological surveillance.

Background Duck circovirus (DuCV) infection in farmed ducks is associated with growth problems or retardation syndromes. Rapid identification of DuCV infected ducks is essential to control DuCV effectively. Therefore, this study aims to develop of an assay for DuCV to be highly specific, sensitive, and simple without any specialized equipment. Methods A set of six specific primers was designed to target the sequences of the Rep gene of DuCV, and A loop-mediated isothermal amplification (LAMP) assay were developed and the reaction conditions were optimized for rapid detection of DuCV. Results The LAMP assay reaction was conducted in a 62°C water bath condition for 50 min. Then the amplification products were visualized directly for color changes. This LAMP assay is highly sensitive and able to detect twenty copies of DuCV DNA. The specificity of this LAMP assay was supported by no cross-reaction with other duck pathogens. Conclusion This LAMP method for DuCV is highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples.

We report a novel electrochemical immunosensor that can sensitively detect avian influenza virus H5 subtype (AIV H5) captured by graphene oxide-H5-polychonal antibodies-bovine serum albumin (GO-PAb-BSA) nanocomposite. The graphene oxide (GO) carried H5-polychonal antibody (PAb) were used as signal amplification materials. Upon signal amplification, the immunosensor showed a 256-fold increase in detection sensitivity compared to the immunosensor without GO-PAb-BSA. We designed a PAb labeling GO strategy and signal amplification procedure that allow ultrasensitive and selective detection of AIV H5. The established method responded to 2(-15) HA unit/50 µL H5, with a linear calibration range from 2(-15) to 2(-8) HA unit/50 µL. In summary, we demonstrated that the immunosenser has a high specificity and sensitivity for AIV H5, and the established assay could be potentially applied in the rapid detection of other pathogenic microorganisms.

A new, rapid, and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR method was developed for simultaneous detection and differentiation of nine avian respiratory pathogens. The respiratory pathogens included in this study were avian influenza subtypes H5, H7, and H9, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophilus paragallinarum (HPG). Ten pairs of primers were designed using conserved and specific sequence genes of AIV subtypes and respiratory pathogens from GenBank. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks of nine respiratory pathogens were observed on the GeXP analyzer. Non-respiratory avian pathogens, including chicken infectious anemia virus, fowl adenovirus, avian reovirus and infectious bursal disease virus, did not produce DNA products. The detection limit for the GeXP-multiplex assay was determined to be 100 copies/μl using various pre-mixed plasmids/ssRNAs containing known target genes of the respiratory pathogens. Further, GeXP-multiplex PCR assay was 100% specific when 24 clinical samples with respiratory infections were tested in comparison with conventional PCR method. The GeXP-multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of nine avian respiratory pathogens.

We report the complete genomic sequence of a novel Newcastle disease virus (NDV) strain, duck/China/Guangxi19/2011, isolated from a white duck in Guangxi Province, southern China. Phylogenetic analysis based on a fusion gene comparison with different NDV strains revealed that duck/China/Guangxi19/2011 is phylogenetically close to genotype IX NDV, and the deduced amino acid sequence of the fusion protein cleavage site was 112R-R-Q-R-R-F117. The whole nucleotide sequence had the highest homology (99.7%) to the sequence of strain F48E8 (GenBank accession number FJ436302). This study will help us understand the epidemiology and molecular characteristics of genotype IX Newcastle disease virus in ducks.

Antibodies specific to the non-structural proteins of viruses are detected in virus-infected animals and show promise as a reliable diagnostic marker for virus infections. We examined the potential use of two non-structural proteins of fowl adenovirus (FAdV)-based, 100K and 33K, enzyme-linked immunosorbent assays (ELISAs) in the diagnosis of FAdVs. We cloned and expressed the 100K and 33K non-structural protein genes of the FAdVs in the pGEX-4T-1 plasmid vector. Purified 100K and 33K proteins alone or in combination were used as antigens in ELISAs. Antibodies specific to the 100K and 33K non-structural proteins were detected in chickens experimentally infected with FAdVs, but not in chickens vaccinated with inactivated FAdVs. In contrast, the agar gel precipitation (AGP) test detected FAdV-specific antibodies in 70.3% of the vaccinated chickens, suggesting that the non-structural protein-based ELISA could be used in the differential diagnosis of infected and vaccinated chickens. To further validate the 100K and 33K-based ELISA (100K-33K-ELISA) method, we compared its sensitivity and specificity with that of a whole virus-based ELISA and an AGP test in detecting FAdV-specific antibodies in 350 field samples. The results showed that the 100K-33K-ELISA exhibited a higher sensitivity than the AGP test and a comparable sensitivity and specificity to the whole virus ELISA. Overall, the 100K-33K-ELISA method is sensitive, specific and can be used to distinguish an acute FAdV infection from an inactivated virus-based vaccination response.

We report here the complete genome sequence of a novel H1N2 avian influenza virus strain, A/Sparrow /Guangxi/GXs-1/2012 (H1N2), isolated from a sparrow in the Guangxi Province of southern China in 2012. All of the 8 gene segments (hemagglutinin [HA], nucleoprotein [NP], matrix [M], polymerase basic 2 [PB2], neuraminidase [NA], polymerase acidic [PA], polymerase basic 1 [PB1], and nonstructural [NS] genes) of this natural recombinant virus are attributed to the Eurasian lineage, and phylogenetic analysis showed that those genes are derived from H1N2, H3N1, H3N2, H4N6, H6N2, H10N8, H5N1, and H4N6 avian influenza viruses (AIVs). The amino acid motif of the cleavage site of HA is PSIQSR↓GLF. The sequence analysis will help in understanding the molecular characteristics and epidemiology of the H1N2 influenza virus in sparrows.

A duplex quantitative real-time polymerase chain reaction (dq-PCR) assay was optimized to simultaneously detect Haplosporidium spp. and Perkinsus spp. of shellfish in one reaction. Two sets of specific oligonucleotide primers for Haplosporidium spp. and Perkinsus spp., along with two hydrolysis probes specific for each parasite group, were used in the assay. The dq-PCR results were detected and analyzed using the Light Cycler 2.0 software system. The dq-PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the dq-PCR assay was 200 template copies for both Haplosporidium spp. and Perkinsus spp. No DNA product was amplified when known DNA from Marteilia refringens, Toxoplasma gondii, Bonamia ostreae, Escherichia coli, Cymndinium spp., Mykrocytos mackini, Vibrio parahaemolyticus, and shellfish tissue were used as templates. A total of 840 oyster samples from commercial cultivated shellfish farms from two coastal areas in China were randomly collected and tested by dq-PCR. The detection rate of Haplosporidium spp. was 8.6 % in the Qindao, Shandong coastal area, whereas Perkinsus spp. was 8.3 % coastal oysters cultivated from shellfish farms of Beihai, Guangxi. The dqPCR results suggested that Haplosporidium spp. was prevalent in oysters from Qindao, Shandong, while Perkinsus spp. was prevalent in oysters from the coastal areas of Beihai, Guangxi. This dq-PCR could be used as a diagnostic tool to detect Haplosporidium spp. and Perkinsus spp. in cultivated shellfish.

Avian reovirus (ARV) is an important pathogen of poultry and causes significant economic losses to the poultry industry. To develop a rapid and sensitive method for the surveillance of ARV, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of six primers specific to the S1 gene segment of ARV. The established assay was performed at 62oC for 60 min in a thermal block, and the result was visualized directly under daylight or ultraviolet light. The detection limit of the RT-LAMP assay was 10 fg total RNA, which was 100-fold higher than that of reverse transcriptase polymerase chain reactions. The specificity of the assay was supported by the lack of cross-reaction with other avian pathogens. Furthermore, viral RNAs of field isolates were successfully detected by the assay. Overall, the newly established RT-LAMP assay is simple, rapid, sensitive, specific, and can visually detect ARV without the use of any specialized equipment.

The complete mitochondrial genome sequence of the Longsheng duck was measured by PCR-based methods. Our research findings revealed that the entire mitochondrial genome of the Longsheng duck was 16,603 bp (GenBank accession number: KJ739616). The contents of A, T, C, and G in the mitochondrial genome were 29.22%, 22.21%, 32.79% and 15.77%, respectively, which were similar to the majority of most avian species. The complete mitochondrial genome of the Longsheng duck contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and one control region. Components of the Longsheng duck’s mitochondrial genome were similar to those of other Anas platyrhynchos in gene arrangement and composition. The complete mitochondrial genome of the Longsheng duck should provide essential information for understanding phylogenetic relationships of duck mitochondrial genome.

House sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Five Newcastle disease virus (NDV) strains were isolated from house sparrows living around the poultry farms in southern China. These isolates were characterized by pathogenic assays and phylogenetic analysis. The results showed that all NDV isolates except one were velogenic and virulent for chickens. These four virulent strains for chickens possess the amino acid sequence (112)R/K-R-Q-K/R-R-F(117) in the F(0) cleavage site which is typical of velogenic NDV. Phylogenetic analysis indicated that these isolates belong to genotype VII and were closely related to the strains which were isolated from NDV outbreaks in chickens since 2000. One isolate of NDV from house sparrow belong to genotype II and was proved to be vaccine strain (Chicken/U.S./LaSota/46). The result of this study proved that house sparrow can carry the virulent NDV strains and the same genotype of viruses that are circulating in poultry are existing in house sparrows living around poultry farm in southern China.

A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) was developed and optimized to simultaneously detect 3 viral pathogens of shrimp. Three sets of specific oligonucleotide primers for Taura syndrome virus (TSV), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) were used in the assay. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 231 bp for TSV, 593 bp for WSSV and 356 bp for IHHNV. No specific bands of the same size were amplified from other penaeid shrimp pathogenic viruses or bacteria. As little as 10 pg of TSV RNA and 100 pg of WSSV DNA and IHHNV DNA could be detected using gel electrophoresis. Studies are in progress to further test the specificity and sensitivity of this mRT-PCR method on viral isolates, as well as on clinical samples.

A multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) was developed and optimized for the detection of type A influenza virus; the assay simultaneously differentiates avian H5, H7 and H9 hemagglutinin subtypes. Four sets of specific oligonucleotide primers were used in this test for type A influenza virus, H5, H7 and H9 heamagglutinin subtypes. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 860 bp for H5, 634 bp for H7, 488 bp for H9 hemagglutinin subtypes, and 244 bp for type A influenza virus. The common set primers for type A influenza virus were able to amplify a 244 bp DNA band for any of the other subtypes of AIV. The mRT-PCR assay developed in this study was found to be sensitive and specific. Detection limit for PCR-amplified DNA products was 100 pg for the subtypes H5, H7, and H9 and 10 pg for type A influenza virus in all subtypes. No specific amplification bands of the same sizes (860, 634 and 488 bp) could be amplified for RNA of other influenza hemagglutinin subtypes, nor specific amplification bands of type A influenza (244 bp) for other viral or bacterial pathogens.