磁珠法测定痕量肝炎病毒的研究

目的寻求灵敏而简便的肝炎病毒临床检测新方法。方法采用氧化硅包覆的磁珠同时提取肝炎病毒的DNA及RNA,结合荧光实时定量聚合酶链反应(PCR)检测乙型肝炎病毒(HBV)及丙型肝炎病毒(HCV)。结果磁珠法可以同时提取HBV-DNA和HCV RNA,磁珠法提取HBV-DNA的效率高于离心柱法和煮沸法,磁珠法提取HCV RNA的效率与离心柱法相近,但提取效率都高于煮沸法。结论将磁珠的富集分离与高灵敏度的检测手段相结合,可显著提高检测的特异性和灵敏度,且方法简便可自动化操作,可以满足大批样品肝炎病毒筛选检测的要求,适用于血液中心筛查及医院检测等领域。     

逆转录套式聚合酶链反应检测庚型肝炎病毒

目的建立敏感、特异的逆转录套式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄ－ＰＣＲ）方法，以检测中国人庚型肝炎病毒（ＨＧＶ）感染。方法根据中国人感染ＨＧＶ者ＮＳ５区部分核苷酸序列分析结果，设计套式ＰＣＲ引物，用于ＨＧＶＲＮＡ的检测，并与用国外报道引物所作的一次ＰＣＲ和套式ＰＣＲ检测结果进行比较。结果共检测标本１３３份。以国外报道引物作一次ＰＣＲ和套式ＰＣＲ检出率分别为８．３％和１１．３％，作者建立的套式ＰＣＲ检出率为１８．０％，对部分ＰＣＲ产物进行序列分析证实为ＨＧＶ特异性基因。结论建立的方法可在中国人群中显著提高ＨＧＶＲＮＡ检出率。     

3种流感病毒检测方法的比较和应用

目的探讨比较荧光定量RT-PCR、普通RT-PCR和细胞培养3种方法在流感病毒检测中的应用,从而确定每种方法各自的实用领域。方法通过荧光定量RT-PCR、普通RT-PCR和狗肾传代细胞(MDCK)培养3种方法检测398件流感样病例(ILI)咽拭子样本。比较3种方法的灵敏度和特异性。结果实时荧光RT-PCR检测阳性样本129件(阳性率为32.4%);普通RT-PCR检测出阳性样本93件(阳性率为23.4%);MDCK细胞培养法分离出流感病毒64株(阳性率为16.1%)。荧光定量RT-PCR的灵敏度达到0.01TCID50,特异性强。结论无论从敏感性、特异性还是从时间上,荧光RT-PCR对于疫情的爆发更有应用价值。细胞培养法分离流感病毒对于核实疫情的实验室诊断和病原学监测具有重大意义。     

Development of an EvaGreen based real-time RT-PCR assay for rapid detection,quantitation and diagnosis of goose calicivirus

An unclassified calicivirus (CV) detected in geese was recently reported and proposed as a new member of the family Caliciviridae. There is limited information about the epidemiology, etiology and detection method of goose-origin CV (GCV) to date. In this study, an EvaGreen based fluorescence quantitative real-time RT-PCR assay was developed and optimized for the detection of GCVs. The assay sensitively detected GCV RNA template with a good linear standard curve. We also demonstrated the specificity and reproducibility of the detection method for GCVs. Thus, the method developed in this study will benefit the investigation of possible sporadic outbreaks of CV infections in geese, as well as epidemiological and etiological studies of GCVs.     

基于离心式微流控技术的直扩PCR法在流感病毒分型检测中的应用

目的 建立一种基于离心式微流控技术的检测流感病毒分型的直扩PCR法。方法 设计特异性引物探针,采用离心式微流控芯片对流感病毒不同分型进行直扩荧光PCR检测,进一步评价该方法灵敏度、特异性、重复性和线性关系;用该直扩荧光PCR法检测576例临床标本,并用核酸检测试剂盒(PCR-荧光探针法)进行验证。结果 离心式微流控直扩PCR法的总体最低检出限为500 copies/mL;与常见呼吸道病原体无交叉反应;精密度均小于4.34%;具有良好的线性关系(R²均>0.98);576例临床标本中检出甲型流感病毒通用型75例,其中H1N1 26例,H3亚型49例;乙型流感病毒通用型74例,均为Victoria系。与对照试剂比较,离心式微流控PCR法在流感病毒不同分型的灵敏度、特异性、总符合率均高于92.9%,两种方法检出阳性率(IAV、H1N1、H1、H3、IBV和IBV-V)差异均无统计学意义(χ²值分别为3.200、0.500、0.500、1.333、2.250、2.250,P均>0.05),具有极好的一致性(Kappa值均>0.96)...     

基于磁珠的ABO血型蛋白质芯片的构建及初步应用分析

目的：构建一种以磁珠为标记物的,用于血型检测的可视化蛋白质芯片。方法用人免疫球蛋白G （IgG）及磁珠标记的抗人IgG进行琼脂糖浓度、点样浓度、温度和固定时间等实验条件的摸索。根据优化的条件,经过铺片、点样、固定、封闭等程序构建ABO血型检测蛋白质微阵列,加入血液样品进行反应,最后用磁珠标记的抗体进行检测。结果通过条件的优化发现,在1．0％的琼脂糖基片表面,以100μg/m L的蛋白质稀释液点样,之后在24℃固定8 h ,能够得到更优的信号强度。检测不同稀释度的抗原抗体,正定型效价可达到1∶8192,反定型可达到1∶4096。检测14例临床样品,结果样点清晰、肉眼可见,且结果判定与试管法一致性达100％。结论成功构建了基于磁珠的血型检测可视化蛋白质微阵列。     

The promise of methylation on beads for cancer detection and treatment

Despite numerous technical hurdles, the realization of true personalized medicine is becoming a progressive reality for the future of patient care. With the development of new techniques and tools to measure the genetic signature of tumors, biomarkers are increasingly being used to detect occult tumors, determine the choice of treatment and predict outcomes. Methylation of CpG islands at the promoter region of genes is a particularly exciting biomarker as it is cancer-specific. Older methods to detect methylation were cumbersome, operator-dependent and required large amounts of DNA. However, a newer technique called methylation on beads has resulted in a more uniform, streamlined and efficient assay. Furthermore, methylation on beads permits the extraction and processing of miniscule amounts of methylated tumor DNA in the peripheral blood. Such a technique may aid in the clinical detection and treatment of cancers in the future.     

免疫纳米荧光碳点技术快速检测乙型副伤寒沙门菌

摘要:目的:建立一种以新型纳米材料——纳米荧光碳点(carbon dots,CDs)免疫示踪技术快速检测样本中细菌的方法。 方法: 以检测乙型副伤寒沙门菌为例建立方法。用抗沙门菌Ha因子抗体耦联CDs制备成免疫纳米荧光碳点;用抗沙门菌O₄因子抗体耦联磁珠粒对样本中乙型副伤寒沙门菌进行富集后,与抗Ha抗体耦联的CDs进行特异性结合,形成抗O₄-磁珠粒-细菌-抗Ha CDs“三明治”式免疫复合物;再用免疫亲和分离液移除复合物中的磁珠粒,借助荧光光谱仪测定分离液中CDs的荧光强度,推算出样品中致病菌的污染情况。 结果：抗Ha-CDs可有效结合抗O₄磁珠粒富集的细菌,对乙型副伤寒沙门菌的最低检测限为10³ CFU/mL,总时间约为2 h。 结论：建立的CDs技术可用于样本中乙型副伤寒沙门菌的快速检测。     

麻疹病毒、风疹病毒和腮腺炎病毒多重RT-PCR法的建立

摘要：目的：建立针对麻疹病毒、风疹病毒和腮腺炎病毒的多重RT-PCR 检测方法。 方法：分别根据麻疹病毒M基因、风疹病毒E基因和腮腺炎病毒M基因设计3 对引物,建立同时检测3种病毒的多重RT-CR,并评估其灵敏度和特异性。 结果：建立的多重RT-PCR可同时或分别扩增的3种病毒的111 bp、352 bp和274 bp基因片段,其灵敏度分别达到2.1、2.1、2.2 lg_{CCID 50}/mL;而与脊髓灰质炎病毒和乙型脑炎病毒无交叉反应。 结论: 建立的多重RT-PCR可用于麻疹病毒、风疹病毒和腮腺炎病毒的快速检测。     

Diagnostic tools for Toscana virus infection

Toscana virus (TOSV; Phlebovirus, Bunyaviridae) is an important etiological agent of acute meningitis and meningoencephalitis in Mediterranean countries. Laboratory diagnosis has been carried out in serological studies using ELISA, immunofluorescence and/or neutralization tests that are not influenced by the virus viability; however, in the acute phase of the infection, nucleic acid amplification techniques are the methods of choice to diagnose viral meningitis from cerebrospinal fluid samples. Molecular methods are rapid and sensitive and, unlike traditional methods, such as virus isolation by cell culture, they are not influenced by the viability of the virus in the clinical specimen; however, the RNA integrity is crucial for the success of these methods. Real-time PCR is the most important molecular method used in laboratories worldwide, since it is less time-consuming and it reduces the risk of contamination. Therefore, a sensitive real-time PCR has been developed for diagnosis of suspected cases of TOSV infection either autochthonous and/or imported, since a new lineage of TOSV, divergent from the Italian prototype, has recently been reported in Spain.     

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus

Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid–-based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02?×?10^?1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.     

呼吸道合胞病毒RNA及IgM在小儿呼吸道感染诊断中的应用价值

目的探讨呼吸道合胞病毒RNA(RSV-RNA)及呼吸道合胞病毒IgM(RSV-IgM)在小儿呼吸道感染诊断中的应用价值。方法选取2015年9月至2017年4月确诊呼吸道合胞病毒感染患儿56例,收集其咽部分泌物标本,对RSV-RNA进行检测;另抽取患儿空腹静脉血,对RSV-IgM进行检测,对两者的阳性检出率及与年龄、发病时间的相关性进行统计学分析。结果 RSV-RNA、RSV-IgM阳性检出率分别为89.29%、32.14%,RSV-RNA的阳性检出率显著高于RSV-IgM,差异有统计学意义(P0.05)。≤6个月,6个月至1岁患儿的RSV-RNA阳性检出率均高于RSV-IgM(P0.05);发病时间≤7d的患儿RSVRNA的阳性检出率高于RSV-IgM(P0.05)。结论在小儿RSV感染检测中,检测RNA比IgM具有更高的准确性,尤其是对年龄≤1岁、发病时间较短(≤7d)的患儿,早期诊断具有重要指导作用。     

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009–10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples.     

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.