NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleic acid sequence-based amplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强.采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当.用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97).     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97)。     

NASBA(依赖核酸序列的扩增)及其在病毒检测中的应用

NASBA(nucleic acid sequence based amplification)即依赖核酸序列的扩增技术,是一种扩增RNA的新技术,是由一对引物介导的、连续均一的、体外特异核苷酸序列等温扩增的酶促反应过程。反应在42℃进行,可以在2h左右将模板RNA扩增约109~12倍,不需特殊的仪器。NASBA已经广泛应用于细菌、病毒等多种病原微生物的检测,就NASBA的技术原理及其在病毒检测中的应用作一综述。     

改良逆转录环介导等温扩增检测技术快速实时检测H9亚型禽流感病毒

利用改良逆转录环介导等温扩增(RT-LAMP)技术建立一种快速、灵敏的检测方法用于H9亚型禽流感病毒检测。根据H9亚型禽流感病毒血凝素(HA)基因保守区序列中的8个区段设计6条特异性引物,在恒温条件下进行核酸扩增反应,并以琼脂糖凝胶电泳和目视检查绿色荧光两种方法对扩增结果进行判定。结果表明,RT-LAMP的最小检测限为100 fg,灵敏度比PT-PCR高100倍,且与H5亚型、H7亚型禽流感病毒,新城疫病毒(NDV)无交叉反应。目视检查绿色荧光与常规琼脂糖凝胶电泳的判定结果一致。整个扩增检测过程在35 min内即可完成。利用临床样本对RT-LAMP法进行验证,结果与RT-PCR一致。由实时浊度分析得到的标准曲线,计算出临床样本中的病毒质粒拷贝数均在2×107-2×104之间。因此,本研究建立的RT-LAMP方法快速、灵敏、特异性强,是H9亚型禽流感病毒的一种高效检测方法。     

轮状病毒NASBA检测研究

轮状病毒是世界范围内流行性胃肠炎暴发的重要病因。以患者粪便为样抽提轮状病毒RNA,在轮状病毒VP7高保守基因区段上设计引物,运用核酸序列依赖的扩增(NASBA)法进行检测,变性琼脂糖凝胶电泳和Northern杂交验证。NASBA预期的特异性产物为392bp,并在仅以目标核酸为模板或在浓度高达1μg/μL的非特异性核酸存在的混合模板中,均有清晰的目标带产生,表现出了很高的特异性。其灵敏度和RT-PCR相同甚至更高,可检测到50pg的核酸,并且当反应时间为3h时检测灵敏度最高。NASBA法扩增效率高、灵敏度高、快速易操作,尤其适用在基层单位推广应用。     

核酸序列依赖性扩增、Real-time PCR及GM试验诊断侵袭性曲霉菌感染的临床应用评价

目的核酸序列依赖性扩增(nucleic acid sequence-based amplification,NASBA)、Real-time PCR及GM试验在侵袭性曲霉菌感染中的诊断价值。方法收集2013年11月~2014年6月临床上曲霉菌感染高危病患的血液标本80例,并根据EORTC/MSG诊断标准分为确诊组8例,拟诊组26例,非感染组46例,分别利用NASBA、real-time PCR及GM试验进行检测,计算3种方法的诊断指标并分析评价。结果 NASBA、real-time PCR及GM试验3种方法的灵敏度分别为76.47%、67.65%、52.94%,特异度分别为80.43%、89.13%、80.43%。联合诊断结果显示,NASBA与real-time PCR串联方案有最好的特异度(100%)及阳性预测值(100%);NASBA与real-time PCR并联方案则最为灵敏(94.12%)。结论 NASBA用于诊断IA最为敏感,而real-time PCR则最为特异,GM试验的表现差于前两者。联合应用的诊断策略,在特定情况下提高临床早期诊断IA的准确性。     

NASBA方法制备HIV／SHIV RNA定量测定标准品及其应用

目的应用NASBA方法制备SIV／SHIV RNA定量测定标准品。方法应用NASBA方法直接扩增SIVmac251病毒gag基因上1476～1685之间的片段,扩增的RNA产物（RS-NASBA）纯化后10倍系列稀释,测定定量曲线、标准曲线,测定该标准品的稳定性和重复性。结果应用Qiagen公司QuantiTect SYBR GREEN RT-PCRKit,该标准品可精确定量到2．033×10 copies／μL。结论外标准品RS．NASBA纯度高,稳定性好,可用于定量测定SIV／SHIV RNA拷贝数。     

双色荧光多重PCR技术及在禽流感病毒检测中的应用

为快速检测7种亚型禽流感病毒,对引物进行双色荧光标记,建立双色荧光多重PCR(DFM-PCR)方法,通过运用DFM-PCR可一次检测出7种禽流感病毒亚型。此方法为应用于病毒或病原等一些要求快速、高通量的检测领域打下基础,而且为禽流感病毒检测以及其他种类病毒等病原检测提供了一种新型方法。     

重组酶聚合酶扩增技术在植物病毒检测中的应用

随着分子生物学技术的发展,多种核酸等温扩增技术逐渐被开发出来。其中,重组酶聚合酶扩增(recombinase polymerase amplification,RPA)作为一种快速、灵敏的检测技术具有很大的优势。目前,RPA已应用于转基因生物、各类病原物及食品安全检测等多个领域,并作为新兴技术在植物病毒检测领域中快速发展。RPA技术只需一对引物,在恒温条件下(37-42℃)只需30 min左右即可完成反应,具有较高的灵敏度与特异性。因此,该技术正迅速成为一种能够用于条件有限的实验室或现场植物病毒检测的手段。本文介绍了RPA技术的检测原理、引物设计和应用方式,综述了其在植物病毒检测中的最新研究进展及存在的问题,为RPA技术在植物病毒检测中的应用提供参考。     

Rapid and sensitive detection of avian influenza virus subtype H7 using NASBA

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.     

Comparison of nucleic acid-based detection of avian influenza H5N1 with virus isolation

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.     

Nucleic acid sequence-based amplification methods to detect avian influenza virus

Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1-H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl's eggs.     

A method to detect major serotypes of foot-and-mouth disease virus

Nucleic acid sequence-based amplification (NASBA) is an isothermal technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A rapid and specific NASBA technique was developed, allowing the detection of foot-and-mouth disease virus genetic material in a range of sample material, including preserved skin biopsy material from infected animals, vaccines prepared from denatured cell-free material, and cell-free antigen-based detection kits. A single pair of DNA oligonucleotide primers was able to amplify examples of all major FMD virus subtypes. The amplified viral RNA was detected by electrochemiluminescence. The method was at least as sensitive as existing cell-free antigen detection methods.     

Evaluation of LightCycler as a Platform for Nucleic Acid Sequence-Based Amplification (NASBA) in Real-Time Detection of Enteroviruses

The nucleic acid sequence-based amplification (NASBA) assay has been demonstrated to be more sensitive for detection of enteroviruses (EV) than RT-PCR. Many laboratories, however, do not have a dedicated instrument for the NASBA assay. This study aimed to evaluate the use of the Roche LightCycler as a platform for performing the NASBA assay for detection of EV. A diverse subgenera of EV were used to assess the specificity of the NASBA assay, including coxsackie, echovirus, poliovirus, and other enteroviruses together with related and unrelated viruses, including rhinovirus, respiratory syncytial virus, herpes simplex virus, adenovirus, influenza virus A, and cytomegalovirus. All species of EV tested were successfully detected using NASBA and no cross reactivity with other viruses was observed. Using serial dilutions of EV to assess sensitivity, the NASBA assay was compared to an in-house EV RT-PCR assay. The NASBA assay demonstrated a higher level of sensitivity. Fifty-one clinical samples positive for EV by viral culture were also evaluated. All NASBA results obtained were concordant with viral culture results. This study confirmed that the NASBA assay for the detection of EV could be readily performed on the LightCycler and easily incorporated into the workflow of a diagnostic laboratory equipped with a LightCycler, thereby eliminating the need for additional instrumentation.     

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Detection of Newcastle disease virus using nucleic acid sequence-based amplification.

Newcastle disease (ND) is a contagious and widespread avian disease affecting most species of birds. ND virus (NDV) is the only member of the avian paramyxovirus serotype 1 (APMV1) causing ND outbreak in bird flocks. The technique of nucleic acid sequence-based amplification (NASBA) is a potential method to rapidly and reliably detect NDV isolates. Here, we describe an effective and unprecedented method for detecting NDV strains of all pathotypes. A conserved region of the fusion protein gene was used for designing oligonucleotides specific to all NDV pathotypes. The dynamic range of this NDV NASBA detection method is comparable to virus culture and therefore the NDV NASBA method is a potential alternative for NDV screening and surveillance.