In recent years,the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination an...In recent years,the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment.Until now,traditional RT-PCR,fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV,but these methods require high-level instruments and experimental conditions,not suitable for the rapid detection in field and farms.In order to develop a rapid,sensitive and practical method to detect and identify AIV subtypes,4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established.Using this method,the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein,without cross reaction with other susceptible avian viruses.In addition,the detection limit of the common H1,H5,H7,and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit),which was 10 times more sensitive than that using the routine RT-PCR.Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR.All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast,convenient and practical method for the clinic test and epidemiological investigation of AIV.展开更多
目的建立快速区分鉴别某种(类)微生物的方法。方法选择几种常用的培养基,将不同的菌株接种在培养基上,通过培养基成分对不同微生物的选择性、显色反应及不同菌株在选择性培养基上具有的特征性菌落形态,初步并快速判定微生物,尤其是致病...目的建立快速区分鉴别某种(类)微生物的方法。方法选择几种常用的培养基,将不同的菌株接种在培养基上,通过培养基成分对不同微生物的选择性、显色反应及不同菌株在选择性培养基上具有的特征性菌落形态,初步并快速判定微生物,尤其是致病类微生物。结果大肠埃希氏菌会与其他致病菌混淆,可以通过结晶紫中性红胆盐(crystal violet neutral red bile salt,VRBA)琼脂将其区分;志贺氏菌在培养基上菌落基本均为半透明,体现的均为培养基本身的颜色;亚硫酸铋(sulfurous acid bismuth,BS)琼脂的选择性较强,但鼠伤寒沙门氏菌在该培养基上长势良好。结论该方法可有效提高鉴别致病菌的效率。展开更多
基金supported by the Special Foundation for State Basic Research Program of China(2013FY113300-8)the National Key R&D Program of China(2016YFD0500800)
文摘In recent years,the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment.Until now,traditional RT-PCR,fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV,but these methods require high-level instruments and experimental conditions,not suitable for the rapid detection in field and farms.In order to develop a rapid,sensitive and practical method to detect and identify AIV subtypes,4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established.Using this method,the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein,without cross reaction with other susceptible avian viruses.In addition,the detection limit of the common H1,H5,H7,and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit),which was 10 times more sensitive than that using the routine RT-PCR.Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR.All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast,convenient and practical method for the clinic test and epidemiological investigation of AIV.
文摘目的建立快速区分鉴别某种(类)微生物的方法。方法选择几种常用的培养基,将不同的菌株接种在培养基上,通过培养基成分对不同微生物的选择性、显色反应及不同菌株在选择性培养基上具有的特征性菌落形态,初步并快速判定微生物,尤其是致病类微生物。结果大肠埃希氏菌会与其他致病菌混淆,可以通过结晶紫中性红胆盐(crystal violet neutral red bile salt,VRBA)琼脂将其区分;志贺氏菌在培养基上菌落基本均为半透明,体现的均为培养基本身的颜色;亚硫酸铋(sulfurous acid bismuth,BS)琼脂的选择性较强,但鼠伤寒沙门氏菌在该培养基上长势良好。结论该方法可有效提高鉴别致病菌的效率。
