双色荧光定量PCR快速分型、定量高危型人乳头状瘤病毒

目的针对高危型人乳头状瘤病毒（humanpapillomavims，HPV）亚型的多样性以及感染病毒载量的高低，旨在建立高危型HPV的定量与分型的快速检测方法，为HPV筛查与治疗提供依据。方法利用2种荧光染料分别标记HPV-16、HPV-33／52／58／67和HPV-18／45、HPV-31探针，同时以B球蛋白基因作为内对照，对120份疑为HPV感染的宫颈脱落细胞样本进行I{I＇V分型与定量，定量范围为5×10^1-5×10^7拷贝/ml，以HC-2杂交捕获法作为“金标准”，评价该方法的特异性与灵敏度。结果荧光定量PCR阳性检出率为52．5％（631120），其中HPV-16、HPV-18／45、HPV-31和m．V-33，52，58，67阳性率分别为36．51％（23／63）、11．11％（7／63）、12．70％（8／63）、58．73％（37／63）；HPV感染的平均病毒载量为1．08×10^7拷贝/ml。荧光定量PCR的特异性为100％，灵敏度为81．82％。结论双色荧光定量PCR能快速分型、定量高危型HPV，可用于HPV感染的筛查与宫颈病变程度预测以及疗效观察。     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

实时荧光PCR直接检测样本中耐甲氧西林金黄色葡萄球菌应用研究

目的 探讨实时荧光PCR直接检测病人样本中耐甲氧西林金黄色葡萄球菌的应用价值.方法 对80例经VITEK2COMPACT药敏鉴定系统鉴定为有金黄色葡萄球菌生长(其中MRSA40例,MSSA40例)及20例未检出金黄色葡萄球菌的患者样本进行实时荧光PCR扩增,检测样本中金黄色葡萄球菌特异性核酸片段(耐热核酸酶基因nuc)及耐药基因(mecA)并与药敏鉴定比较,判断实时荧光PCR法检测MRSA的敏感性及特异性;对于两种方法检测结果不一致的18例样本重新接种培养,收集金黄色葡萄球菌菌株进行实时荧光PCR扩增,检测nuc基因及mecA基因,以期发现两种方法检测结果差异原因.结果 100例临床样本两种方法检测结果一致共82例,其中MRSA一致40例,MSSA一致22例,非金葡20例;两种方法检测结果不一致18例,均为PCR基因检测为MRSA而药敏鉴定为MSSA.MRSA检测药敏鉴定阳性40例,检出率为40％,PCR基因检测阳性58例检出率为58％;PCR基因检出率明显高于药敏鉴定,二者结果差异有显著性(P＜0.05);以药敏鉴定为标准,PCR基因检测MRSA的敏感性为100％,特异性为70％;18例金黄色葡萄球菌菌株实时荧光PCR检测MRSA结果与药敏鉴定一致.结论 实时荧光PCR直接检测患者样本中MSSA具有高度敏感性及特异性,但检测MRSA会受到样本中其他含有mecA基因菌的干扰,对于临床结果解释需慎重.即便如此,该方法对于MRSA检测在院感防控方面仍有重要意义.     

鸭乙型肝炎病毒cccDNA Taq Man荧光定量PCR的建立

目的建立鸭乙型肝炎病毒（DHBV）cccDNA TaqMan荧光定量PCR方法。方法用pUCm-T载体和PCR纯化产物连接，转染TOP10菌，筛选阳性菌落，提取质粒，制作外标准品；在DHBV基因组负链两侧设计一对引物，并在引物间设计和荧光标记一段TaqMan探针，严格优化反应物的组成和扩增条件，建立了DHBVcccDNA TaqMan荧光定量PCR方法。结果最低可检出104拷贝／ml；批内误差为0．2％～3．14％，批间误差为2．22％～4．43％；在10^5—10^12拷贝／ml之间与Ct值具有很好的线性[Ct=-2．8361ln（x）＋41．45，r=-0．9985]。结论该法是一种灵敏度高、特异性强、较为简便的DHBV cccDNA定量检测技术。     

α变形菌纲磁螺菌属TaqMan探针实时荧光定量PCR快速检测方法的建立

目的建立一种快速有效检测环境中q变形菌纲磁螺菌属细菌的方法。方法本研究以α变形菌纲磁螺菌属保守且特异的16SrRNA片段为靶序列,化学合成基因序列,制备重组质粒作为磁螺菌属检测的标准品;用实时荧光定量聚合酶链反应（FQ—PCR）技术,针对目的片段基因设计特异性引物和TaqMan荧光探针,进行实时荧光定量PCR检测,运用统计学方法评价该方法的特异性、敏感性及重复性。结果本实验成功构建了质粒PUC19-QC,引物、探针特异性良好,标准曲线在5．10×10^1～5．10×10^7拷贝数之间具有较好的线性关系,相关系数R2=0．999。该法最低可检测到5．10x10个DNA拷贝数,灵敏度明显高于普通PCR;重复性试验表明．荧光定量PCR检测结果Ct值波动范围较小,α值的标准差均小于0．23,表明该法重复性好,可靠性高。析因方差分析表明不同稀释度之间的ct值差异有统计学意义（F=3125．305,P〈0．001）,三个批次的再现性良好,差异无统计学意义（F=0．057,P=0．945）,而浓度分组与时间之间也无交叉效应（F=0．533,P=0．873）。结论本实验所建立的TaqMan探针实时荧光定量PCR检测技术,能够快速有效地检测仅变形菌纲磁螺菌属细菌,且检测结果稳定,受外界条件如时间、气温等影响小。     

单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立

目的 建立一种快速、特异、灵敏、准确定量的单核细胞增生(单增)李斯特菌(Listeriamonocytogenes)与志贺菌(Shigella)同步检测方法.方法 分别根据单增李斯特菌溶血素O基因hly与志贺菌侵袭性质粒抗原H基因ipaH设计合成引物和探针.构建重组质粒pGEM-T-hly与pGEM-T-ipaH,并以EcoR I单酶切使环状重组质粒线性化作为标准品.优化反应体系,分析特异性.双重荧光定量PCR对人工污染的脱脂灭菌乳进行检测.结果 成功构建了重组质粒标准品,并运用5'、3'端分别标记FAM、TAMRA的hly基因探针和5'、3'端分别标记HEX、TAMRA的ipaH基因探针成功建立了单增李斯特菌与志贺菌同步荧光定量PCR检测方法.结论 建立的方法有较强的特异性,线性范围好(105～101copies/μl,R2≥0.998),灵敏度为10 copies/PCR,同步检测人工污染脱脂灭菌乳的灵敏度为102CFU/ml.     

一种新型冠状病毒荧光定量PCR快速检测方法的应用评价

目的:建立并评价一种基于COYOTE ? Flash20实时荧光定量PCR仪用于新型冠状病毒(SARS-CoV-2)核酸快速检测的方法。 方法:通过使用靶向SARS-CoV-2 ORF1ab和N基因的特异性引物探针组构建快速反应体系,并验证体系的灵敏度和特异性。同时,使用108例新型冠状病毒肺炎(...     

登革热病毒RNA实时荧光定量RT-PCR检测方法的建立

目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒（DV）及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。     

实时荧光定量PCR法筛查唐氏综合征的实验研究

目的建立一种无创性、高特异性的快速筛查唐氏综合征（Down’s syndrome，DS）的检测方法。方法采用实时荧光定量PCR技术对正常人和DS患者外周血样本的有核细胞进行检测，根据△Ct值的差异建立检测方法。结果正常组和唐氏组△Ct值有显著性差异（P〈0．001），初步建立了实时荧光定量PCR筛查唐氏综合征的快速检测方法。结论实时荧光定量PCR具有无创性、特异性高且简单、快速等优点，适用于唐氏综合征产前筛查。     

双重荧光定量RT—PCR在快速检测A、B型流感病毒上的应用

目的 建立双重荧光定量RT-PCR技术同时快速检测A、B型流感病毒,并应用于临床样本的检测.方法 在A型流感病毒M基因和B型流感病毒HA基因的保守区序列分别设计特异性引物和Taqman探针,建立优化双重荧光RT-PCR反应体系,评价所建双重RT-PCR反应体系的特异性、敏感性和稳定性,并应用于疑似流感含漱液标本检测.结果 该方法对A、B型流感病毒检测具有高度特异性,检出限分别为0.1TCID50和0.01TCID50,具有较好的稳定性.可从疑似流感患者含漱液中直接检测到流感病毒核酸.结论 本研究建立的双重荧光定量RT-PCR可以同时准确分型A、B型流感病毒,灵敏度高,稳定性好,是一种快速检测流感病毒的新方法.     

Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report results, and there may be difficulty in rapidly and accurately identifying methicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were validated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n = 106), methicillin-sensitive S. aureus (n = 134), and coagulase-negative Staphylococcus (n = 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory.     

Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) by using selective broth and real-time PCR (broth-PCR) was developed and evaluated. The samples (n = 304) were cultured in the broth overnight, followed by nuc gene detection by real-time PCR. nuc-negative samples were further checked for the presence of nuc amplification inhibitors by a PCR internal inhibitor assay. nuc-positive samples and nuc-negative samples with PCR inhibitors were cultured onto plates and processed further. The diagnostic values for this MRSA screening method were 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. The application of the broth-PCR method will be able to report most of the negative samples (258 of 289 [89.3%]) on the next morning and can save as much as 84.9% (258 of 304) of the labor and cost spent on processing the nuc-negative specimens on plates. In the study, all the samples were processed in parallel by the broth enrichment method and the plating method for comparison. To identify MRSA, the isolated oxacillin-resistant S. aureus strains were tested by a duplex real-time PCR targeting the mecA gene and the nuc gene. A collection of MRSA, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and methicillin-susceptible Staphylococcus epidermidis strains and a panel of standard strains of 11 bacterial species other than S. aureus were also tested by this method, which was proved to be a valuable tool for MRSA identification in a routine microbiological laboratory.     

Real-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.     

Gram stain-specific-probe-based real-time PCR for diagnosis and discrimination of bacterial neonatal sepsis

Sepsis is a serious disease with high mortality in newborns. It is very important to have a convenient and accurate method for pathogenic diagnosis of neonatal sepsis. We developed a method of simultaneous detection and Gram classification of clinically relevant bacterial pathogens causing sepsis directly from blood samples with Gram stain-specific-probe-based real-time PCR (GSPBRT-PCR). With GSPBRT-PCR, 53 clinically important strains representing 25 gram-positive and 28 gram-negative bacterial species were identified correctly with the corresponding Gram probe. The limits of the GSPBRT-PCR assay in serial dilutions of the bacteria revealed that Staphylococcus aureus could be detected at concentrations of 3 CFU per PCR and Escherichia coli at concentrations as low as 1 CFU per PCR. The GSPBRT-PCR assay was further evaluated on 600 blood specimens from patients with suspicion of neonatal sepsis and compared to the results obtained from blood cultures. The positive rate of the GSPBRT-PCR array was 50/600 (8.33%), significantly higher than that of blood culture (34/600; 5.67%) (P = 0.00003). When blood culture was used as a control, the sensitivity of GSPBRT-PCR was 100%, the specificity was 97.17%, and the index of accurate diagnosis was 0.972. This study suggests that GSPBRT-PCR is very useful for the rapid and accurate diagnosis of bacterial infection and that it can have an important impact on the current inappropriate and unnecessary use of antibiotics in the treatment of newborns.     

New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci

Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.     

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.     

Molecular probes for diagnosis of clinically relevant bacterial infections in blood cultures

Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided into Pseudomonas species, Pseudomonas aeruginosa, Escherichia coli, and other Gram-negative species. Within the Gram-positive species, probes were designed for Staphylococcus species, Staphylococcus aureus, Enterococcus species, Streptococcus species, and Streptococcus pneumoniae. The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targeting Pseudomonas spp., P. aeruginosa, E. coli, Streptococcus spp., S. pneumoniae, Enterococcus spp., and Staphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific for S. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.     

Multiplex PCR for Simultaneous Identification of Staphylococcus aureus and Detection of Methicillin and Mupirocin Resistance

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.     

Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10³ CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.     

Real-time PCR assay for detection of fluoroquinolone resistance associated with grlA mutations in Staphylococcus aureus

Resistance to fluoroquinolones among clinical isolates of Staphylococcus aureus has become a clinical problem. Therefore, a rapid method to identify S. aureus and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of S. aureus and mutations at codons Ser-80 and Glu-84 of the grlA gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-S. aureus bacterial species tested. A total of 85 S. aureus isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the S. aureus isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of S. aureus and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of S. aureus and its susceptibility to fluoroquinolones.