SYBR Green实时PCR检测恶性疟原虫和间日疟原虫的临床检验技术研究

目的建立鉴别恶性疟和间日疟的SYBR Green实时PCR检测方法。方法针对恶性疟原虫和间日疟原虫18S rRNA基因设计引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的SYBR Green实时PCR,并进行54例临床标本检测,其中恶性疟32例、间日疟22例。以镜检法为金标准分析敏感度和特异度等指标。结果该方法可扩增出恶性疟原虫和间日疟原虫18S rRNA基因片段,并能检出混合感染,特异性好。该方法检测恶性疟原虫或间日疟原虫的敏感度为97.30%(36/37),特异度为5.88%(1/17);检测恶性疟原虫,敏感度为87.50%(21/24),特异度为63.33%(19/30);检测间日疟原虫,敏感度为69.23%(9/13),特异度为68.29%(28/41)。结论所建立的SYBR Green实时PCR方法能较准确地诊断疟疾并鉴别虫种,敏感度高,在混合感染的诊断方面具有优越性。     

百日咳鲍特菌BP283基因的实时荧光定量PCR检测方法的建立

目的探讨检测百日咳鲍特菌BP283基因片段的有效方法。方法以百日咳鲍特菌BP283序列为目的基因,设计探针和引物,构建质粒标准品;建立FQ-PCR体系,优化反应条件,并进行方法学评价。结果成功构建了重组质粒pCR2.1-BP283;建立了检测BP283基因片段的FQ-PCR方法:标准曲线相关系数为0.998,最低检测浓度为102copies/μL,对临床其他常见呼吸道病原体不出现特异性扩增曲线,批内及批间变异系数均<4%。结论FQ-PCR方法可成功检测百日咳鲍特菌BP283基因,该方法具有敏感性好、特异性高及重复性好等优点。     

Human herpesvirus 6 (HHV-6) isolation from bone marrow: HHV-6- associated bone marrow suppression in bone marrow transplant patients [see comments]

These studies tested the hypothesis that human herpesvirus 6 (HHV-6) can cause posttransplant bone marrow (BM) suppression in BM transplant (BMT) patients. Fifteen adult patients who received T-lymphocyte- depleted, allogeneic BM transplants and who developed posttransplant BM suppression were studied. Detailed chart reviews were used to divide the patients into two groups: (1) those with diagnosed BM suppression (DBMS) and (2) those with idiopathic BM suppression (IBMS). BM aspirates obtained from the patients at the onset of BM suppression were subjected to an HHV-6 isolation procedure using mitogen-stimulated blood mononuclear cells. BM specimens obtained from another population of BMT patients solely to document engraftment irrespective of their BM function were also subjected to the HHV-6 isolation procedure as controls. HHV-6 was isolated from 6 of 15 BM samples from BMT patients with BM suppression. BM samples from patients with IBMS were more likely to be positive for HHV-6 than those from patients with DBMS (P < .01). Also, HHV-6-positive BM were significantly more likely (P < .05) to come from patients with suppression of more than one BM lineage than HHV-6-negative BM. Finally, samples of BM from an unselected series of BMT patients studied without regard to their BM function were less likely (P < .01) to be positive for HHV-6 than patients with IBMS.     

Quantitation of human herpesvirus-6 (HHV-6) DNA in a cord blood transplant recipient with chromosomal integration of HHV-6

Chromosomal integration of the human herpesvirus-6 (HHV-6) genome (CIHHV-6) is an important consideration if HHV-6 DNA is detected during the course of transplantation. A 4-year-old girl with refractory anemia with excess blasts type-2 was diagnosed with CIHHV-6 before a cord blood transplantation. HHV-6 DNA was serially quantitated by polymerase chain reaction assay in the transplant period. The possibility of HHV-6 reactivation in a transplant recipient with CIHHV-6 was suspected in our case.     

Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis

Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.     

Low prevalence of antibody to human herpesvirus-6 (HHV-6) in Kadazans

The prevalence of antibody to human herpesvirus type 6 (HHV-6) and Epstein-Barr virus (EBV) viral capsid antigens (VCA) were analysed in sera from Kadazans of Sabah, North Borneo. At a serum dilution of 10, about 34% were positive for HHV-6 antibody but in contrast all 95 individuals studied were positive for EBV VCA antibody. The study shows that HHV-6 and EBV infection occur independently. The low frequency of seropositive individuals in this community suggests that other than socioeconomic factors are responsible for the spread of the virus.     

Management of CMV, HHV-6, HHV-7 and Kaposi-sarcoma herpesvirus (HHV-8) infections in patients with hematological malignancies and after SCT

These recommendations were prepared by the European Conference on Infections in Leukaemia following a predefined methodology. Literature searches were made to identify studies pertinent to management of CMV, HHV-6, -7 and -8 infections. For CMV, 76 studies were reviewed: 72 published and 4 presented as abstracts. Twenty-nine of these studies were prospective randomized trials. For the other herpesviruses, HHV-6, -7 and -8, no randomized controlled trial has been performed, although data from some studies with other primary endpoints have been used to assess the management of HHV-6 infection. Works presented only as abstracts were used to a very limited extent. The quality of evidence and level of recommendation were graded according to the Center for Disease Control (CDC) criteria.Bone Marrow Transplantation (2008) 42, 227-240; doi:10.1038/bmt.2008.162; published online 30 June 2008.     

Indirect immunofluorescence and real time PCR for detection of Simkania negevensis infection in Danish adults with persistent cough and in healthy controls

Simkania negevensis is a recently discovered intracellular organism that has been associated with respiratory tract infections. To determine the seroprevalence of the organism in adult Danes and to study the association between the organism and persistent cough, we developed an immunofluorescence assay based on S. negevensis infected Hep2 cells for antibody determination and a real time PCR assay for direct detection of the organism. Among 100 healthy blood donors, 41 (41%) had IgG antibodies to S. negevensis (cut-off titre =1:16) and the antibody level increased with increasing age (correlation coefficient 0.124, p=0.037). 80 of 185 patients (43%) with chronic cough had IgG antibodies to S. negevensis which was no different from the 41% in the control population (Chi2=0.13, p=0.72). None of the patients or controls had any detectable IgA antibodies to S. negevensis. PCR was performed on nasopharyngeal aspirates from a subgroup of 176 patients with persistent cough and in none of these was S. negevensis DNA detected. We conclude that the seroprevalence of S. negevensis is high in Denmark and positively correlated with age. However, we were unable to show an association between S. negevensis and persistent cough.     

Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences.     

Real‐time RT‐PCR assays for type and subtype detection of influenza A and B viruses

Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of ‘type,’ i.e., influenza virus A and B, and ‘subtype,’ i.e., H1, H3, and H5, specific, single‐step/reaction vessel format, real‐time RT‐PCR assays using total RNA from archived reference strains, shell‐vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. ‘Type’ and ‘subtype’ specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field‐deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.     

检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立

目的 建立SYBR Green Real-time PCR检测和筛选黄病毒属病毒方法. 方法 参照文献报道的通用引物,以JEV cDNA 和DEN cDNA为模板,建立RT-PCR和SYBR Green Real-time PCR,检测和筛选黄病毒属病毒,并比较两者的敏感性. 结果 此黄病毒属引物适合两种反应体系,SYBR Green Real-time PCR方法的敏感性是RT-PCR方法的100倍,最低检出病毒浓度为0.5×10-2 PFU/ml. 结论 建立的两种方法均可用于黄病毒属病毒检测,以SYBR Green Real-time PCR方法具有更高的敏感性,对于黄病毒属病毒初筛检测具有应用价值.     

空肠弯曲菌hipO基因的Real-time PCR检测研究

目的建立基于新型Taqman荧光探针的荧光实时(Real-time)PCR方法用于空肠弯曲菌的筛选检测与快速鉴定。空肠弯曲菌(Campylobacter jejuni)是近十几年来在世界范围内广泛重视的人畜共患病原菌,可以导致人类的急性肠炎与食物中毒,并能引发格林-巴利综合征等并发症。为更好地利用分子生物学技术对空肠弯曲菌进行快速、准确的基因检测,本研究从样品增菌液与单菌落中提取细菌DNA,建立了针对空肠弯曲菌特异的hipO(hippuricase,马尿酸酶)基因的Real-time PCR方法,用于空肠弯曲菌的快速筛选检测与可疑菌落快速鉴定。试验结果表明,该方法检测细菌的灵敏度为1~10CFU,人工布菌鸡肉的检测灵敏度为1~10CFU。本研究所建立的空肠弯曲菌荧光实时PCR方法具有准确、可靠、快速的特点。