人血浆DNA双重实时荧光定量PCR检测法的建立

目的 建立带有内参照的双重实时荧光定量PCR方法检测血浆DNA含量.方法 构建重组质粒DNA作为内参照物,采用共用下游引物的双重实时荧光定量PCR技术同步扩增人看家基因β-actin和重组质粒载体中人工合成DNA序列,定量检测健康成年人血浆DNA含量.结果 本法能在同一个反应管中对目的基因和内参照进行同步扩增,两者的扩增无相互干扰,特异性好;重组质粒DNA的平均扩增效率达90%,β-actin基因的平均扩增效率接近100%;本法批内变异系数（CV）11%,批间CV17%.结论 成功建立含有内参照的双重实时荧光定量PCR方法,可对血浆DNA进行定量检测.     

The performance of the HPV16 real-time PCR integration assay

OBJECTIVES: Integration of high risk human papillomavirus (HPV) into the host genome leads to viral oncogene deregulation predisposing to neoplastic progression. Integration can be detected from pap smear or biopsy and use as marker of progressive disease. DESIGN AND METHODS: We have previously developed a highly sensitive real-time PCR method to determine HPV integration frequency and viral load of HPV16 in clinical samples. The test is accurate and sensitive detecting approx. 50 copies of integrated HPV in the sample. RESULTS: We found that a tenfold excess of episomal form to integrated form interferes with the test, regardless the amount of viral DNA. The same was true with background DNA more than 1500 ng in reaction. CONCLUSIONS: Overall, this method is reproducible and suitable for high-throughput screening of clinical samples, but excess episomal copies might mask the integrated form.     

多重PCR法和HPV分型基因芯片法在高危型人乳头瘤病毒感染检测中的应用

目的评价高危型人乳头瘤病毒（HPV）多重核酸扩增荧光检测法和HPV分型基因芯片检测法在HPV感染女性患者标本基因分型的临床应用效果。方法应用13种高危型HPV多重PCR荧光检测法对在深圳市人民医院进行宫颈癌筛查的653例疑似HPV感染女患者的宫颈细胞样本进行检测,并与HPV分型基因芯片检测法的检测结果进行比较,2种方法检测13种高危型HPV不一致的样本经序列分析方法进一步验证。结果13种高危型HPV多重PCR荧光检测法检测HPV阳性样本,阳性检出率为21．5％（140／653）;用HPV分型基因芯片检测法验证,与13种高危型HPV多重PCR荧光检测法一致的阳性样本占20．4％（133／653）,总一致率为98．2％。2种方法的检测结果具有高度一致性（kappa值一o．945）;用HPV分型基因芯片检测法检出：HPV单一型别感染占59．4％（79／133）,主要的高危HPV型别为HPV16、52、39、68、33和59型,6种高危型占总数的87．3％（69／79）,其中HPV16和HPV52为主要感染,占44．9％。结论高危型HPV多重PCR荧光检测法和HPV分型基因芯片检测法在13种高危型HPV的检结果具有高度一致性;多重PCR荧光检测法覆盖主要的13种高危型HPV,分型基因芯片检测法可进行具体的单一型别分型。2种检测方法的联合应用,对宫颈癌筛查和预防具有较高的临床应用价值,同时可为HPV分子流行病学和HPV疫苗的应用研究提供依据。     

Multicolour,multiplex real-time PCR assay for the detection of human-pathogenic poxviruses

After eradication of variola virus, one of the most dangerous infectious diseases affecting mankind, today other poxviruses of different genera can cause infection in humans. These viruses include human-specific molluscipoxviruses as well as zoonotic orthopoxviruses and parapoxviruses. While non-variola orthopoxvirus infections mostly cause mild symptoms in immunocompetent persons, they can evoke severe disease in immunocompromised patients. Since the typical poxviral skin lesions are rarely diagnosed by physicians, PCR-based identification of suspected poxviruses is often required. To simplify the PCR-based diagnosis of human-pathogenic poxviruses, we established a multicolour multiplex real-time PCR that simultaneously detects and differentiates human-pathogenic poxviruses in one reaction. Using 5′ nuclease probes labelled with FAM for orthopoxviruses, VIC for parapoxviruses and FAM and VIC for molluscipoxviruses, respectively, amplification of poxviral DNA resulted in a genus-specific reporter-dye profile. Validation with 36 human clinical specimens and DNA of pathogens causing pox-like skin lesions demonstrated the specificity of the assay. Probit analysis revealed a limit of detection of 9.7, 22.08 and 28.1 copies/assay (95% CI) for molluscipoxvirus, orthopoxvirus and parapoxvirus DNA, respectively. The combinatorial multicolour strategy applied has the potential to be used in further applications targeting even more than three pathogens.     

1型HIV感染多重PCR检测方法的建立

重复性,能覆盖我国最主要的流行病毒株.     

实时荧光定量PCR反应检测宫颈脱落细胞人乳头瘤病毒16/18型和巨细胞病毒的协同感染

目的探讨宫颈脱落细胞人乳头瘤病毒16/18型和巨细胞病毒协同感染的关系。方法采用实时荧光定量PCR反应(FQ-PCR)同时检测两种病毒的感染率。结果38例宫颈癌中检出HPV16/18型35例(阳性率92.11%),CMV11例(阳性率28.95%)。32例宫颈癌前病变(CIN)中检出HPV16/18型20例(阳性率62.50%),CMV7例(阳性率21.87%)。30例正常宫颈脱落细胞中检出HPV16/18型5例(阳性率16.67%),未检出CMV。结论宫颈脱落细胞HPV16/18型感染率随宫颈病变程度的加重而升高,且差异有显著性(P<0.05),CMV感染率随宫颈病变程度的加重而升高但差异无显著性(P>0.05),CMV仅是协同HPV16/18型感染导致宫颈癌的发生。     

HPV病毒检测联合液基细胞学和鳞状细胞癌抗原检测在筛查宫颈癌中的价值

目的探讨HPV病毒阳性患者联合应用液基细胞学检测(TCT)、鳞状细胞癌抗原检测(SCCAg)检测在筛查宫颈癌中的价值。方法 1125例常规门诊体检HPV病毒阳性的患者,按照1:1:1比例随机分为三组(TCT组、SCC-Ag组和TCT+SCC-Ag组),所有患者行阴道镜检查并取活检,以病理诊断为金标准,计算各组筛查宫颈癌的灵敏度、特异度、阳性预测值和阴性预测者,评价各方法的诊断价值。结果 TCT联合SCC-Ag筛查宫颈癌的敏感度、特异度、阳性预测值、阴性预测者分别为0.68、0.96、0.89和0.85,与单独TCT筛查宫颈癌相比,有明显优越性,差异有统计学意义(P0.05)。结论 HPV病毒检测联合TCT和SCC-Ag明显降低筛查宫颈癌的假阳性率,提高特异度和诊断符合率,在宫颈癌筛查中有推广价值。     

Simple multiplex real-time PCR for rapid detection of common 16S rRNA methyltransferase genes

We have developed a real-time multiplex PCR assay to detect the three most common 16S rRNA methyltransferase genes (armA, rmtB and rmtC), which encode problematic high-level resistance to all clinically-relevant aminoglycoside antibiotics. All results were consistent with published conventional PCR assays and these genes still appear rare in Australia.     

实时荧光定量-聚合酶链反应方法检测肺炎支原体

目的 建立一种快速、灵敏、特异的肺炎支原体实时荧光定量-聚合酶链反应(real-time PCR)检测方法,以期用于临床肺炎支原体感染检测。 方法 通过测序分析和序列比对,选取肺炎支原体p1基因中保守区域设计特异性引物和荧光探针,建立和完善此real-time PCR检测方法,并进行扩增效率、灵敏度及特异度评价。与已报道的肺炎支原体常规聚合酶链反应(PCR)方法进行150份临床标本检测能力比较。 结果 建立的real-time PCR方法对肺炎支原体的检测限约为10 cfu。使用该方法对9株肺炎支原体ATCC标准株和30株临床分离株核酸扩增均为阳性;对10种其他支原体、13种常见呼吸道病原菌染色体及人类染色体扩增结果均为阴性。同时,临床标本的检测结果显示该方法检测灵敏度优于常规PCR。 结论 本研究建立的real-time PCR方法可快速、灵敏、特异地检测标本中肺炎支原体核酸,可适用于临床肺炎支原体诊断。     

A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers

Objectives

Deletion polymorphisms of Glutathione-S-transferase (GST) M1 and T1 are considered risk factors for various diseases. However, most previous studies only distinguished “null” and “non-null” genotypes. Our aim was to develop a reliable, high-throughput GSTM1/T1 genotyping method able to determine allele copy numbers.

Design and methods

We developed a multiplex real time PCR method to distinguish between heterozygous (1/0) and homozygous (1/1) GSTM1 and GSTT1 genotypes. The principle of relative quantification was applied and an expectation-maximisation (EM) algorithm was developed to assign one of 3 possible genotypes: 1/1, 1/0 or 0/0 for each of the two genes.

Results

1320 Caucasians were genotyped using the newly developed method. The observed genotype distributions did not deviate from the expected and were in Hardy-Weinberg equilibrium. GSTM1 duplication was detected in one sample.

Conclusion

This new semiquantitative genotyping method is a sensitive and promising tool for large-scale molecular epidemiological and clinical studies.     

HPV16/18感染及其致癌基因与宫颈癌的关系

目的研究端粒酶活性、人乳头瘤病毒16/18(HPV16/18)感染及致癌基因与宫颈癌的相关性。方法对30例浸润型宫颈癌、58例宫颈内瘤形成(CINⅢ20例、CINⅡ20例、CINⅠ18例)及16例正常女性的宫颈新鲜组织,采用端粒酶重复序列扩增法(TRAP-PCR)检测端粒酶活性,用荧光定量多聚酶链式反应(FQ-PCR)检测HPV16/18DNA,用PCR方法对HPV16/18DNA阳性组织作HPV16型致癌基因E6、E7检测。结果宫颈癌组中端粒酶阳性22例(73.33%)(HPV16/18+15例,7例有E6、E7表达),端粒酶阴性8例(HPV16/18+1例,无E6、E7表达)。CINⅢ级中端粒酶阳性13例(46.4%)(HPV16/18+6例,2例有E6、E7表达),端粒酶阴性15例(无HPV16/18+);CINⅡ级中端粒酶阳性6例(25%)(HPV16/18+2例,1例有E6、E7表达),端粒酶阴性18例(无HPV16/18+);CINⅠ级中端粒酶阳性2例(10.00%)(HPV16/18+2例,无E6、E7表达)。而16例正常宫颈组织仅1例(6.25%)端粒酶活性表达,此例HPV也呈阳性而无E6、E7表达。宫颈癌和癌前病变(CINⅢ)组织端粒酶活性表达频率(P<0.01)和HPV16/18感染率(P<0.01)及其致癌基因表达(P<0.01)均显著高于良性损伤(CINⅠ和CINⅡ)和正常对照。结论端粒酶活性在宫颈癌发生中可能起到重要作用,在子宫颈损伤中端粒酶激活与HPV感染及其致癌基因的表达密切相关;对于探讨宫颈病变的进展和宫颈癌的发生具有重要意义。     

HPV16／18感染及其致癌基因与宫颈癌的关系

目的 研究端粒酶活性、人乳头瘤病毒16／18（HPV16／18）感染及致癌基因与宫颈癌的相关性。方法 对30例浸润型宫颈癌、58例宫颈内瘤形成（CINⅢ20例、CINⅡ20例、CINⅠ18例）及16例正常女性的宫颈新鲜组织，采用端粒酶重复序列扩增法（TRAP-PCR）检测端粒酶活性，用荧光定量多聚酶链式反应（FQ-PCR）检测HPV16／18DNA，用PCR方法对HPV16／18DNA阳性组织作HPV16型致癌基因E6、E7检测。结果 宫颈癌组中端粒酶阳性22例（73．33％）（HPV16／18＋15例，7例有E6、E7表达），端粒酶阴性8例（HPV16／18＋1例，无E6、E7表达）。CINⅢ级中端粒酶阳性13例（46．4％）（HPVl6／18＋6例，2例有E6、E7表达），端粒酶阴性15例（无HPV16／18＋）；CINⅡ级中端粒酶阳性6例（25％）（HPV16／18＋2例，1例有E6、E7表达），端粒酶阴性18例（无HPV16／18＋）；CINⅠ级中端粒酶阳性2例（10．00％）（HPV16／18＋2例，无E6、E7表达）。而16例正常宫颈组织仅1例（6．25％）端粒酶活性表达，此例HPV也呈阳性而无E6、E7表达。宫颈癌和癌前病变（CINⅢ）组织端粒酶活性表达频率（P〈0．01）和HPV16／18感染率（P〈0．01）及其致癌基因表达（P〈0．01）均显著高于良性损伤（CINⅠ和CINⅡ）和正常对照。结论 端粒酶活性在宫颈癌发生中可能起到重要作用，在子宫颈损伤中端粒酶激活与HPV感染及其致癌基因的表达密切相关；对于探讨宫颈病变的进展和宫颈癌的发生具有重要意义。     

弗氏枸橼酸杆菌TaqMan实时荧光定量-聚合酶链反应检测方法的建立

目的 建立针对弗氏枸橼酸杆菌的TaqMan实时荧光定量-聚合酶链反应(real time-PCR)检测方法。 方法 针对弗氏枸橼酸杆菌的特有序列设计引物和TaqMan探针,扩增目的基因建立标准曲线,确定检测方法的灵敏度;对20种其他肠道致病菌及院内感染中常见的致病菌进行检测,评价该检测方法的特异性;使用牛奶模拟标本评价方法在实际检测工作中应用性。 结果 TaqMan real time-PCR检测方法对弗氏枸橼酸杆菌重组质粒的检测灵敏度为1.0101拷贝／反应体系;该检测方法在检测30种其他肠道致病菌及院内感染中常见的致病菌时未出现特异性扩增。该检测方法对牛奶模拟样本中弗氏枸橼酸杆菌检测下限为1.0102cfu/ml的菌量;通过对1.0107、1.0105和1.0103三个浓度质粒标准品的重复检测,确定本方法的组内变异系数为1.90%~3.91%;组间变异系数为1.52%~1.69%。 结论 本研究建立的TaqMan real time-PCR检测方法可作为检测弗氏枸橼酸杆菌灵敏、特异、快速的方法。     

HPV DNA和HPV E6/E7 mRNA检测在宫颈癌筛查中的应用价值探讨

目的探讨HPV DNA和HPV E6/E7 mRNA检测在宫颈癌筛查中的应用价值。方法选取宫颈病变筛查的受试者407例为研究对象,均行宫颈薄层液基细胞学(TCT)检查,并检测细胞学标本中HPV DNA和HPV E6/E7 mRNA的表达。215例行阴道镜宫颈活检,结合组织病理诊断结果进行统计分析。结果 407例细胞病理学诊断结果:正常范围(WNL)192例(47.17%)、意义不明的不典型鳞状上皮细胞(ASCUS)106例(26.04%)、低度鳞状上皮内病变(LSIL)70例(17.20%)和高度鳞状上皮内病变(HSIL)39例(9.58%)。诊断为LSIL和HSIL患者中,HPV DNA和HPV E6/E7 mRNA检出率差异无统计学意义(P0.05);诊断为ASCUS患者中,HPV DNA检出率显著高于HPV E6/E7 mRNA(P0.01)。215例组织病理学诊断结果:正常109例,阴性106例,包括CINⅠ级44例、CINⅡ级27例、CINⅢ级32例、宫颈鳞癌3例。对于诊断为CINⅡ级、CINⅢ级/宫颈癌患者,HPV DNA和HPV E6/E7 mRNA检测的灵敏度、特异度和阳性预测值差异无统计学意义(P0.05),但HPV E6/E7 mRNA检测的阴性预测值和准确度显著高于HPV DNA检测(P0.05或P0.01)。结论 HPV E6/E7 mRNA检查较HPV DNA检查能更好地评估HPV感染患者宫颈病变恶化风险,联合TCT检测对提高宫颈癌早期筛查和诊断的准确度具有积极意义。     

Rapid assay for detection of methicillin-resistantStaphylococcus aureususing multiplex PCR

The presence or absence of themecAgene, the determinant of resistance to all β-lactam antibiotics, was examined in clinical isolates ofStaphylococcus aureusby multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bpnuc-based PCR fragment (amplification product of thenucgene encoding specificStaphylococcus aureusnuclease) and a 533-bpmecA-based PCR fragment (amplification product of themecAgene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.     

A novel attention-guided convolutional network for the detection of abnormal cervical cells in cervical cancer screening

Early detection of abnormal cervical cells in cervical cancer screening increases the chances of timely treatment. But manual detection requires experienced pathologists and is time-consuming and error prone. Previously, some methods have been proposed for automated abnormal cervical cell detection, whose performance yet remained debatable. Here, we develop an attention feature pyramid network (AttFPN) for automatic abnormal cervical cell detection in cervical cytology images to assist pathologists to make a more accurate diagnosis. Our proposed method consists of two main components. First, an attention module mimicking the way pathologists reading a cervical cytology image. It learns what features to emphasize or suppress by refining extracted features effectively. Second, a multi-scale region-based feature fusion network guided by clinical knowledge to fuse the refined features for detecting abnormal cervical cells at different scales. The region proposals in the multi-scale network are designed according to the clinical knowledge about size and shape distribution of real abnormal cervical cells. Our method, trained and validated with 7030 annotated cervical cytology images, performs better than the state of art deep learning-based methods. The overall sensitivity, specificity, accuracy, and AUC of an independent testing dataset with 3970 cervical cytology images is 95.83%, 94.81%, 95.08% and 0.991, respectively, which is comparable to that of an experienced pathologist with 10 years of experience. Besides, we further validated our method on an external dataset with 110 cases and 35,013 images from a different organization, the case-level sensitivity, specificity, accuracy, and AUC is 91.30%, 90.62%, 90.91% and 0.934, respectively. Average diagnostic time of our method is 0.04s per image, which is much quicker than the average time of pathologists (14.83s per image). Thus, our AttFPN is effective and efficient in cervical cancer screening, and improvement of clinical workflows for the benefit of potential patients. Our code is available at https://github.com/cl2227619761/TCT_Detection.     

Comparative evaluation of a rapid MRSA detection assay based on multiplex real-time PCR versus MRSA screening cultures containing egg yolk

Although the Centers for Disease Control and Prevention (CDC) has been recommending the performance of active surveillance culture (ASC) for the prevention of methicillin-resistant Staphylococcus aureus (MRSA) infections and their control since the guideline was issued in 2006, many variant types of MRSA with various characteristics have been found recently. As this change in MRSA characteristics makes it harder to screen MRSA only by cultures, it is expected that ASC will not be sufficient for the prevention of MRSA infections or MRSA infection control. We evaluated the comparative utility of the BD GeneOhm MRSA assay (a rapid MRSA detection test based on a multiplex real-time polymerase chain reaction [PCR] assay; Becton Dickinson, Fukushima, Japan) and MRSA screening cultures containing egg yolk. The results of the BD GeneOhm MRSA assay were as follows: all MRSA strains were positive, including one strain which became positive by retest; all methicillin-resistant S. epidermidis (MRSE) strains and methicillin-sensitive S. epidermidis (MSSE) strains were negative; 11 of 12 methicillin-sensitive S. aureus (MSSA) strains were negative, while 1 strain was positive; ATCC 33591 was positive, and ATCC 29213 was negative. The sensitivity and specificity of the BD GeneOhm MRSA assay were 100% and 97.4%, respectively. Nine egg yolk reaction-negative MRSA strains were found in 50 MRSA strains, and all MRSE, MSSA, and MSSE strains were denied as MRSA on Pourmedia MRSA II (Eiken Chemical, Tokyo, Japan) after 24-h or 48-h incubation at 35°C. The sensitivity and specificity of Pourmedia MRSA II were 82% and 100%, respectively. Similar results were obtained with some other cultures containing egg yolk.     

Comparison of real-time PCR and MassTag PCR for the multiplex detection of highly pathogenic agents

Multiplex PCR assays are a cost- as well as labour-effective way to analyse one sample for several pathogens simultaneously. Besides the mutual competition of the individual PCR reactions included in a multiplex PCR assay, their specific read-out displays a limiting factor for the total number of PCR reactions that can be multiplexed. In this study, two PCR systems with different read-out approaches are compared, using a pentaplex PCR assay for the detection of highly pathogenic agents. A pentaplex assay was used since five represents the current limit of real-time PCR multiplexing capacity due to the low resolution of fluorescence emission peaks of the current equipment. In contrast, MassTag PCR as a quite new technique offers the possibility to detect up to 20-30 target sequences from one reaction. After extensive and separate optimisation of the PCR protocol for both platforms, a comparative probit analysis showed good sensitivities for MassTag and real-time PCR detection. Nevertheless, the detection limits of MassTag PCR have been undercut by the real-time PCR for each target. We therefore conclude that MassTag PCR is a useful diagnostic technique for the sensitive screening for pathogens by highly multiplexed PCR assays, but cannot reach the sensitivity of real-time PCR for lower multiplexed PCR assays.