烟草环斑病毒的定量检测技术研究

目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。     

呼吸道合胞病毒TaqMan探针实时定量RT-PCR检测方法的建立及应用

目的:建立呼吸道合胞病毒(RSV)核酸特异、快速、敏感的TaqMan探针实时荧光定量PCR检测方法,并对临床样本进行检测。方法:比对编码RSV非编码蛋白的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量RT-PCR检测方法,并与传统RT-PCR方法进行比较,分别对两者的灵敏性、特异性、重复性及临床样本检验的适用性进行评价。结果:所建立的实时荧光定量RT-PCR检测方法可用于RSV的特异性检测。相对于传统RT-PCR方法100拷贝/反应的检测灵敏度,实时荧光定量RT-PCR的检测灵敏度达到10拷贝/反应,检测范围为1010~101拷贝/反应,且具有良好的特异性和重复性。从169份临床呼吸道标本中检出RSV阳性40例,高于普通PCR方法(31/169)。结论:建立了RSV的TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,在临床上具有较好的应用前景。     

寨卡病毒、登革病毒、基孔肯雅病毒三重荧光定量RT-PCR检测方法的建立及评价

本研究旨在建立寨卡病毒(ZIKV virus,ZIKV)、登革病毒(Dengue virus,DENV)以及基孔肯雅病毒(Chikungunya virus,CHIKV)三种病毒快速筛查、诊断的核酸检测技术。选用ZIKV的NS1基因、DENV的NS5蛋白基因以及CHIKV的E1蛋白基因作为靶标区域设计三组特异性引物探针,建立三重实时荧光定量RT-PCR检测方法。用ZIKV、DENV、CHIKV病毒体外转录RNA和病毒细胞培养物对该方法的灵敏性、特异性、重复性等方面进行评价,最后临床样本以及模拟标本验证。结果显示:三重实时荧光定量RT-PCR检测方法扩增效率均可达到90%以上,三种病毒体外转录RNA最低检测限均低于15拷贝/PCR,病毒培养物最低检出限均低于10PFU/mL且与单重检测方法无明显差异。与其他病毒无交叉反应,变异系数均在2%以内。临床标本及模拟标本检出率均可达95%以上。本研究建立的检测寨卡病毒、登革病毒以及基孔肯雅病毒的三重实时荧光RT-PCR方法具有良好的敏感性、特异性和重复性,可用于寨卡病毒病等相关临床标本的检测。     

实时荧光定量PCR及RT-PCR与常规PCR及RT-PCR检测对虾病毒效果的比较

常规PCR及RT-PCR已用于对虾DNA及RNA病毒检测,但存在费时、灵敏度较低、不能定量等问题。建立了TaqMan实时荧光定量PCR及RT-PCR方法,分别用于检测白斑综合症病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)及桃拉综合征病毒(TSV)、黄头病毒(YHV)4种对虾病毒。与常规PCR及RT-PCR比较,所建立的TaqMan实时荧光定量PCR及RT-PCR检测上述4种对虾病毒不仅有很高的特异性,检测灵敏度也提高了10～100倍,同时还具有快速、简便、不污染环境、重复性好、实时定量等优点,可明显提高对虾病毒检验检疫工作质量及效率。     

TaqMan—MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒。针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析。结果表明:引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应。方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好。本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测。     

人偏肺病毒TaqMan-MGB探针实时定量RT-PCR检测方法的建立及初步应用

目的:建立人偏肺病毒(hMPV)核酸特异的快速、敏感的TaqMan-MGB探针实时定量RT-PCR检测方法.方法:分别设计hMPV特异的引物与荧光标记探针,合成hMPV绝对定量RNA模板,建立实时荧光定量PCR方法,并与常规RT-PCR平行比较,对其灵敏性、特异性和可重复性,以及用于临床样本的适用性等进行评价.结果:本方法可对hMPV进行特异性诊断,检测灵敏度可达10拷贝/25 μL,检测线性范围至少可达10 1～10 6拷贝/反应,且实验重复性好,初步应用于北京地区采集的158份临床鼻咽拭子标本,定量RT-PCR检出31份标本阳性,明显高于常规RT-PCR方法(22/158).结论:建立了人偏肺病毒TaqMan-MGB探针定量RT-PCR检测方法,并初步证实可用于临床鼻咽拭子标本的检测,为开展hMPV的流行监测及临床早期诊断提供了技术手段.     

应用改良的实时TaqMan荧光定量RT-PCR技术检测口蹄疫病毒及其3D基因转录水平

将改良的实时TaqMan荧光定量RT-PCR技术应用于口蹄疫病毒感染体内和体外的定量检测以及其3D基因转录水平分析。结果表明:对样品中口蹄疫病毒基因组RNA的检测灵敏度可达l0个基因拷贝,可同时检测病毒正负链复制水平且重复性较好,所测口蹄疫病毒3D基因转录水平可高达6.9×104拷贝/μL;与实时SYBRGreenⅠ染料RT-PCR技术比较,改良的实时TagMan荧光定量RT-PCR技术检测灵敏度高6.7倍。以上结果证实,改良的实时TaqMan荧光定量RT-PCR技术在病毒检测和基因表达水平分析方面有更高的灵敏度和特异性。     

TaqMan-MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒.针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析.结果表明引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应.方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好.本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测.     

细胞培养结合实时荧光RT-PCR法快速检测甲肝病毒滴度的研究

目的建立一种细胞培养与实时荧光RT-PCR相结合的快速检测甲肝病毒滴度的方法。方法根据甲肝病毒(HAV)L-A-1株5'端基因组序列,设计了2条基因特异性引物及一条探针,建立实时荧光RT-PCR法,结合细胞培养检测甲肝病毒滴度,并与ELISA检测法进行比较。结果实验中建立的方法能特异检测甲肝病毒,细胞培养8d检测病毒滴度为lg107.0CCID50/mL。同一样本重复检测3次,批内样本Ct值的变异系数最大为0.89%,批间样本Ct值变异系数最大为1.66%。建立的细胞培养结合实时荧光RT-PCR法(细胞培养8 d)与细胞培养ELISA法(细胞培养28 d)检测甲肝病毒滴度结果差异无统计学意义(P0.05)。结论该方法具有快速、灵敏、特异等优点,应用于疫苗常规检测有良好前景。     

甲型H1N1流感病毒核酸荧光定量RT-PCR检测技术

目的:建立基于TaqMan荧光探针技术的甲型H1N1流感病毒实时定量RT-PCR方法。方法:分析H1N1流感病毒基因特性,根据新发甲型H1N1流感病毒突变基因片段设计检测引物和TaqMan荧光探针,建立荧光定量RT-PCR检测体系,体外转录制备RNA标准品,进行特异性、敏感性、重复性及盲样检测评价实验。结果:可特异性有效检测新发甲型H1N1流感病毒核酸,与H1～H16流感病毒基因无交叉反应;对RNA标准品的检测敏感性达103拷贝/μL;重复性实验中,阳性标准品Ct值变异系数(CV)10%,阴性标准品检测结果均呈阴性;12份盲样检测结果特异性好。结论:该荧光定量RT-PCR方法可作为甲型H1N1流感防控的病原快速诊断技术。     

Using a graph of the abnormal predictive value versus the positive predictive value for the determination of outlier laboratories in the National Health Service cervical screening programme

R.G. Blanks Using a graph of the abnormal predictive value versus the positive predictive value for the determination of outlier laboratories in the National Health Service cervical screening programme. Objective: The positive predictive value (PPV) for the detection of cervical intraepithelial neoplasia (CIN) grade 2 or worse of referral to colposcopy from moderate dyskaryosis or worse (equivalent to high‐grade squamous intraepithelial lesion or worse) is a standard performance measure in the National Health Service cervical screening programme. The current target is to examine ‘outlier’ laboratories with PPVs outside the 10th–90th percentile, which automatically identifies 20% of laboratories for further investigation. A more targeted method of identifying outliers may be more useful. Methods: A similar measure to the PPV, the abnormal predictive value (APV), can be defined as the predictive value for CIN2 or worse for referrals from borderline (includes atypical squamous and glandular cells) and mild dyskaryosis (equivalent to low‐grade squamous intraepithelial lesion) combined. A scatter plot of the APV versus the PPV can be produced (the APV‐PPV diagram). Three kinds of ‘outlier’ can be defined on the diagram to help determine laboratories with unusual data. These are termed a true outlier value (TOV) or an extreme value (EV) for either PPV or APV, or a residual extreme value (REV) from the APV‐PPV best line of fit. Results: Using annual return information for 2007/8 from 124 laboratories, two were defined as having EVs for PPV (both had a relatively low PPV of 62%). For APV, four laboratories were considered to have EVs of 34%, 34%, 34% and 4% and one was considered to be a TO with an APV of 45%. Five were identified as REV laboratories, although three of these were also identified as having extreme or outlier values, leaving two that had not been identified by the other methods. A total of eight (6%) laboratories were therefore identified as meriting further investigation using this methodology. Conclusions: The method proposed could be a useful alternative to the current method of identifying outliers. Slide exchange studies between the identified laboratories, particularly those at opposing ends of the diagram, or other further investigations of such laboratories, may be instructive in understanding why such variation occurs, and could therefore potentially, lead to improvements in the national programme.     

猪细小病毒LDR-PCR检测方法的建立和应用

为准确特异灵敏地检测猪细小病毒(PPV),建立一种新的LDR-PCR方法。首先在病毒的保守区内设计一对LDR探针,LDR探针两端各连有一段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。以标准质粒为模板,通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,可以特异地检测PPV,与猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)无交叉反应;最低检测限为102个拷贝。利用建立的方法对41例临床样本进行检测,14份样品PPV阳性,与普通PCR检测结果符合率为97.6%。     

Evaluation of Four Commercial Multiplex Molecular Tests for the Diagnosis of Acute Respiratory Infections

Acute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden’s index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86–1.00; PPV:78.95–100.00; Sp:97.32–100.00] & B [YI:0.44–1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50–0.99; PPV:85.71–100.00; Sp:99.38–100.00], hMPV [YI:0.71–1.00; PPV:83.33–100.00; Sp:99.37–100.00], EV/hRV [YI:0.62–0.82; PPV:93.33–100.00; Sp:94.48–100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20–0.80; PPV:57.14–100.00; Sp:98.14–100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections.     

A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician’s skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as “Patho-tb”, for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen’s kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 10³ cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.     

Interaction of Plum Pox Virus with Specific Colloidal Gold-Labeled Antibodies and Development of Immunochromatographic Assay of the Virus

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in sam- ples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by “sandwich”-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.     

猪圆环病毒2型、猪繁殖与呼吸综合征病毒及猪细小病毒混合感染的流行病学调查

根据GenBank上发表的PRRSVORF7、PPVVP2及PCV的基因组序列设计合成引物,建立了分别用于检测PRRSV、PPV和PCV的RT-PCR、PCR及复合PCR方法。应用建立的复合PCR方法对送检的127份病料进行了PCV的检测,对鉴定为PCV2阳性的67份病料再分别进行PRRSV和PPV的检测,以确定猪群中PCV2与PRRSV和,或PPV混合感染情况,结果表明,35份样品表现为PRRSV与PCV2混合感染,占样品总数的52．3％;18份样品表现为PCV2与PPV混合感染,占26．9％。另外,还有一定比例的三重感染,共5个样品,占7．5％。由此可见,猪群中PCV2与PRRSV及PPV混合感染比较普遍。     

Simple and Robust Realtime QRS Detection Algorithm Based on Spatiotemporal Characteristic of the QRS Complex

The purpose of this research is to develop an intuitive and robust realtime QRS detection algorithm based on the physiological characteristics of the electrocardiogram waveform. The proposed algorithm finds the QRS complex based on the dual criteria of the amplitude and duration of QRS complex. It consists of simple operations, such as a finite impulse response filter, differentiation or thresholding without complex and computational operations like a wavelet transformation. The QRS detection performance is evaluated by using both an MIT-BIH arrhythmia database and an AHA ECG database (a total of 435,700 beats). The sensitivity (SE) and positive predictivity value (PPV) were 99.85% and 99.86%, respectively. According to the database, the SE and PPV were 99.90% and 99.91% in the MIT-BIH database and 99.84% and 99.84% in the AHA database, respectively. The result of the noisy environment test using record 119 from the MIT-BIH database indicated that the proposed method was scarcely affected by noise above 5 dB SNR (SE = 100%, PPV > 98%) without the need for an additional de-noising or back searching process.     

Characterization of a Novel Porcine Parvovirus Tentatively Designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca²⁺ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A₂ (PLA₂) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.     

Arterial Pressure Variation as a Biomarker of Preload Dependency in Spontaneously Breathing Subjects – A Proof of Principle

Objective

Pulse (PPV) and systolic pressure variation (SPV) quantify variations in arterial pressure related to heart-lung interactions and have been introduced as biomarkers of preload dependency to guide fluid treatment in mechanically ventilated patients. However, respiratory intra-thoracic pressure changes during spontaneous breathing are considered too small to affect preload and stroke volume sufficiently for the detection by PPV and/or SPV. This study addressed the effects of paced breathing and/or an external respiratory resistance on PPV and SPV in detecting preload dependency in spontaneously breathing subjects.

Methods

In 10 healthy subjects, hemodynamic and respiratory parameters were evaluated during progressive central hypovolemia (head-up tilt). Breathing conditions were varied by manipulating breathing frequency and respiratory resistance. Subjects responding with a reduction in stroke volume index ≥15% were classified as having developed preload dependency. The ability for PPV and SPV to predict preload dependency was expressed by the area under the ROC curve (AUC).

Results

A breathing frequency at 6/min increased the PPV (16±5% vs. 10±3%, p<0.001) and SPV (9±3% vs. 5±2%, p<0.001) which was further enhanced by an expiratory resistance (PPV: 19±3%, p = 0.025 and SPV: 10±2%, p = 0.047). These respiratory modifications, compared to free breathing, enhanced the predictive value of PPV with higher accuracy (AUC: 0.92 vs. 0.46).

Conclusion

Under conditions of progressive central hypovolemia, the application of an external respiratory resistance at a breathing frequency of 6/min enhanced PPV and SPV and is worth further study for detection of preload dependency from arterial pressure variations in non-ventilated subjects.