Development And Serises Of RT-LAMP Methods For Detection Of Avian Influenza Viruses

The influenza A virus genome is composed of eight single-stranded RNA segments that frequently reassortant when two different viruses infect the same host. On the basis of differences in the antigenicity of the two surface glycoproteins, HA and NA, influenza A viruses are categorized into different subtypes. Currently,17HA subtypes and10NA subtypes have been identified. All subtypes were identified initially from avian species, except for the H17N10subtype, which was found in bats. Outbreaks of avian influenza result in significant economic losses to the poultry industry and posing a serious threat to human health. Among the different subtypes of avian influenza, only the H5and H7subtypes are highly pathogenic to birds and are therefore referred to as high-pathogenic avian influenza (HPAI). Infections with these viruses typically result in100%mortality in chickens and are identified as class A infectious diseases by the World Organization for Animal Health. In recent years, infections caused by influenza virus subtype H7have been observed frequently. For example, in1999-2000, the H7N1virus outbreak in Italy led to the deaths of more than13million chickens, causing the extensive economic losses. In2003, an H7N7subtype influenza outbreak in the Netherlands not only impacted the poultry industry but also infected89people, demonstrating that these viruses pose a threat to public health and poultry industries worldwide. A novel reassortant avian influenza A (H7N9) virus emerged and spread among humans in China in March2013. There have been over300people infected with H7N9virus, and over50of these cases resulted in death. Rapid diagnosis and timely monitoring of potential avian influenza outbreaks are among the first important steps in the prevention and control of avian influenza. Currently, several methods are available for the detection of avian influenza. Among the conventional detection methods, virus isolation and molecular biological characterization are the most feasible and accurate techniques. However, the isolation and identification of viruses require extended periods of time ranging from days to weeks; therefore, this method does not meet the time requirements needed for the prevention of epidemics. In addition, high-pathogenic avian influenza viruses pose the potential danger of infecting various mammal species; therefore, these viruses must be handled in biosafety level3facilities. However, most quarantine facilities do not possess these containment capabilities. Therefore, rapid, sensitive and specific molecular biological techniques have played important roles in the rapid detection of avian influenza viruses. Therefore, rapid, sensitive and specific molecular biological techniques, including RT-PCR, and real-time RT-PCR and IFA have played important roles in the rapid detection of avian influenza viruses. Nevertheless, all of these techniques require sophisticated instrumentation (such as PCR machines), limiting the effectiveness of these procedures in smaller, under-equipped laboratories. The loop-mediated isothermal amplification (LAMP) technique is a molecular biology method used to amplify specific DNA fragments in vitro. This method does not require an additional reverse transcription step, and only a water bath is needed to amplify large amounts of nucleic acids. LAMP is therefore a suitable method for smaller laboratories or as an on-site rapid diagnostic tool. In this study, we develop a serial of Reverse Transcription Loop-Mediated Isothermal Amplification Method based on the M, HA and NA genes for the Rapid Detection of Avian Influenza Virus.1. Development of a RT-LAMP method for detection of avian influenza virusA series of specific primer were designed based on the6different regions of M gene sequence of avian influenza virus (AIV)(A/Goose/Guangdong/1/96(H5N1)). A reverse transcriptase loop-mediated isothermal amplification (M-RT-LAMP) for the rapid detection of AIV was developed by using these specific primers. Different avian influenza viruses can be effectively detected by this method. The detection limit of the M-RT-LAMP method reached to0.01PFU AIV, and to be10-fold higher than that of the routine RT-PCR. The amplification could be finished within30min with good specificity and high sensitivity. With the addition of Fluorescent Detection Reagent, the presence of the M-AIV could be detected by naked eyes.2. Development of a RT-LAMP method for detection of subtype H9avian influenza virusA series of specific primer were designed on the hemagglutinin (HA) gene sequence of H9subtype avian influenza virus (H9-AIV)(A/Chicken/HuNan/33/2008(H9N2)), targeting the6distinct sequences of gene. A reverse transcriptase loop-mediated isothermal amplification (H9-RT-LAMP) for the rapid detection of H9-AIV was developed by using the specific primers. The detection limit of the H9-RT-LAMP method reached to0.01PFU H9-AIV, and to be100-fold higher than that of ordinary the One-step RT-PCR. The amplification could be finished within30min with good specificity and high sensitivity. Six different H9-AIV isolates isolated recently years can be effectively detected by this method. The virus can be effectively detected from samples collected from chickens artificially infected by H9-AIV by the H9-RT-LAMP and virus isolation at1day post infection. While the virus can be detected by the routine RT-PCR at3days post infection. These results demonstrated that the H9-RT-LAMP method can be useful to the early diagnosis of avian influenza virus.3. Development of a RT-LAMP method for detection of subtype H5avian influenza virusA series of specific primer were designed based on the hemagglutinin (HA) gene sequence of H5subtype avian influenza virus (H5-AIV)(A/Goose/Guangdong/1/96(H5N1)), targeting the6distinct sequences of gene. A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) for the rapid detection of H5-AIV was developed by using the specific primers. The detection limit of the H5-RT-LAMP method was0.1PFU per reaction for the H5-AIV, and to be100-fold higher than that of routine one-step RT-PCR. The amplification could be finished within30min with good specificity and high sensitivity. Different H5-AIVs isolated form chicken, duck and pigs can be effectively detected by this method.4. Development of a RT-LAMP method for detection of subtype H7N9avian influenza virus A series of specific primer were designed on the hemagglutinin (HA) gene sequence of H7subtype avian influenza virus (H7-AIV)(A/Chicken/HeB/2/2002(H7N2)), targeting the6distinct sequences of gene. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7avian influenza virus (H7AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was0.1-0.01PFU per reaction for H7AIV RNA, making this assay100-fold more sensitive than the routine RT-PCR method. This RT-LAMP assay also has the capacity to detect both high-and low-pathogenic H7AIV strains. The minimum detection limit of the H7-RT-LAMP assay was0.01PFU per reaction for the H7N9influenza virus that was isolated from various sources. These results indicated that the H7-RT-LAMP assay could be usedfor the detection of H7N9influenza viruses. The virus can be effectively detected from samples collected from chickens artificially infected by H7-AIV by the H7-RT-LAMP and virus isolation at1day post infection. While the virus can be detected by the routine RT-PCR at3days post infection. These results demonstrated that the H7-RT-LAMP method can be much sensitive and be useful to the early diagnosis and epidemiological surveillance of avian influenza virus subtype H7.A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9virus. The minimum detection limit of the RT-LAMP assay was0.01PFU H7N9virus, making this method100-fold more sensitive to the detection of the H7N9virus than conventional RT-PCR. The H7N9virus RT-LAMP assays can efficiently detect different sources of H7N9influenza virus RNA (from chickens, pigeons, the environment and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets of Shanghai and Anhui province, as well as human H7N9virus, in less than30min. These results suggest that the H7N9virus RT-LAMP assays were efficient, practical and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples.