食品污染产单核李斯特菌不同检测方法学评估

目的评价以不同方法检测食品中污染产单核李斯特菌（LMO）效果，并对其检测方法进行优化。方法同时用国标法、改良国标法、改良分子信标-实时荧光PCR法、免疫磁性分离法对深圳市2004—2006年食品样品进行LMO分离鉴定并进行比较。结果228份样品中8份增菌液LMO实时荧光PCR阳性，其中6份LMO细菌培养阳性。国标法、改良国标法，改良分子信标-实时荧光PCR法、免疫磁性分离法都能栓出LMO，栓出率分别为2、63％、2．63％、3．50％、2、63％。深圳市食品中LMO的污染率为2.63％（国标法），速食类冻品污染率为12．8％（国标法）。结论荧光PCR法栓出率最高，将检测时间由原来的至少4—5d缩短至1d，可用于食品污染LMO状况调查的初筛扣食物中毒的快速诊断；国标法与改良国标法的栓出率相等，而后者的操作更方便。免疫磁性分离法的栓出率最低。     

几种CMV相关物检测对诊断肾移植患者CMV感染的评价

目的 比较血中pp65抗原、DNA和IgM抗体检测对肾移植患者巨细胞病毒（CMV）活动性感染的诊断价值,为临床上对CMV感染的诊断和治疗提供参考.方法 收集83位肾移植患者的血标本（共302份）,分离血浆和多型核白细胞,血浆用于CMV-DNA（定性PCR）和IgM抗体（ELISA）检测,白细胞用于pp65抗原血症检测（荧光免疫法）.结果 CMV-DNA阳性率为36.1%,CMV IgM抗体阳性率20.2%,CMV pp65抗原阳性率为38.5%.pp65抗原阳性细胞数与病人的临床症状密切相关.结论 pp65抗原阳性细胞数能反应CMV的数量,可监测早期活动性CMV感染及疗效观察,检测方法特异,灵敏,操作简单,适合临床实验室应用.     

我国首次发现的HIV-2型病毒的分子生物学证据

「目的」从分子生物学角度进一步证实我们发现的１例ＨＩＶ感染者为国内首例ＨＩＶ－２型病毒携带才。「方法」从Ｇｅｎｅｂａｎｋ查询已知的ＨＩＶ－２核酸序列,通过序列比较以及酶切位点分析查找ＨＩＶ－２基因组中保守区,应用套式ＰＣＲ扩增出ＨＩＶ－２基因组ＬＴＲ－ＧＡＧ区的￣３３０ｂｐ的核酸片段,并经限制怀酶切位点和序列分析证实扩增片段为ＨＩＶ－２特异性片段。「结果」实验表明,扩增片段为ＨＩＶ－２特异性片段,     

Reliability of gender determination using the polymerase chain reaction (PCR) for single cells

Contamination with extraneous DNA sequences is a frequent problem when performing PCR analysis of single cells. This report describes our experience with eliminating contaminating DNA sequences from PCR reagents for the purposes of gender identification. We have used amplification of Y-specific sequences to identify the gender of single human amniocytes. Female cells consistently showed no Y-specific bands but only 80% of male cells showed the expected intense Y-specific band. This phenomenon could lead to incorrect gender identification of single cells. We developed a technique of simultaneous amplification of X- and Y-specific sequences to prevent misdiagnosis because of failed PCR, which allows accurate preimplantation gender determination for women at risk for conceiving children with X-linked genetic discoses. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment     

Identification of a new humannm23 gene,nm23-H3b

Nm23 is a kind of effective tumor metastasis suppressor genes which included two types in human:nm23-H1 andnm23-H2. Amino acid identity betweennm23-H1 andnm23-H2 was 88%. In this study, using a pair of primers to flank the part of coding sequence ofnm23, the 5′-translated sequence was amplified by polymerase chain reaction (PCR) from human normal liver genomic DNA. A 375-base pairs clone was characterized, which designatedpnm23-H3b. Thenm23-H3b nucleotide sequence between 40 bp and 70 bp is different fromnm23-H1 andnm23-H2, and other sequences have 86% and 90% identical tonm23-H1 andnm23-H2, respectively. Southern blot containingBgl I-digested human liver genomic DNA hybridized to the entirenm23-H3b DNA and showed three bands at 10.5, 7.9 and 4.0 Kb. These data demonstrate that the third humannm23 exists possibly. Therefore,nm23 may be considered a family of closely related genes.     

Diagnosis of tuberculosis by polymerase chain reaction

For the diagnosis of extrapulmonary tuberculosis in adults and all forms of tubercular infections in children, microscopic and cultural techniques have (seen shown to be inadequate. Many serological techniques have been employed for non culture diagnosis of tuberculosis. Early promising results have repeatedly given way to subsequent findings of non-specificity. Major mycobacterial antigens have been shown to be heat shock proteins which are highly conserved in nature. DNA probes for tuberculosis are specific but have a sensitivity equivalent to AFB smear examination. Polymerase Chain Reaction (PCR) with its ability to selectively amplify DNA fragments of interest offers a potentially powerful technique for the rapid, specific and sensitive diagnosis of tuberculosis. Samples from partially treated patients could be culture negative but can be detected by PCR.     

Previous hydatidiform mole identified as the causative pregnancy of choriocarcinoma following birth of normal twins

For appropriate clinical management of patients with gestational trophoblastic tumors it is important to ascertain both the nature of the causative pregnancy and the time interval between that pregnancy and the diagnosis of the tumor. It has been shown that the immediately antecedent pregnancy may not be the causative pregnancy in some cases of choriocarcinoma, particularly where there is a history of molar pregnancy. We report further studies of a case where the causative pregnancy was shown to be a hydatidiform mole, not the immediately antecedent normal term pregnancy. We describe the use of the polymerase chain reaction (PCR) to amplify short tandem repeat polymorphisms in DNA prepared from pathologic blocks of the patient's previously recognised molar pregnancy. A comparison of these polymorphisms with those in the parental and tumor DNA has enabled us to confirm that this hydatidiform mole was indeed the causative pregnancy. Molecular genetic techniques provide a rapid method of determining whether a choriocarcinoma is gestational and, if so, identifying the causative pregnancy.     

Implications of human papillomavirus type for survival in cervical squamous cell carcinoma

In a Swedish series of 107 invasive squamous carcinomas of the cervix, DNA extraction from paraffin-embedded material was successful in 97 cases. The prevalence of human papillomavirus (HPV) in this material was 86.6%, as determined by polymerase chain reaction (PCR) using both consensus and type-specific primers. HPV type 16 was most common (42.3%; other types were 31 (12.3%), 18 (9.3%) and 33 (10.3%). Seventeen cases (17.3%) were positive for the consensus primers only and were regarded as HPV of unknown type. There was no significant difference in corrected survival between patients with HPV-positive or -negative tumors. In the HPV-positive group, patients with tumors containing HPV 33 or HPV 18 had a significantly poorer prognosis than patients with tumors containing other types of HPV DNA (relative hazard 3.18, 95% confidence interval 1.37–7.39, P = 0.007), implying a prognostic significance of HPV type.     

Identification of a Novel Receptor Tyrosine Kinase Expressed in Acute Myeloid Leukemic Blasts

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.     

Molecular evidence for central nervous system involvement in children with newly diagnosed acute lymphoblastic leukemia

Central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) would have profound implication for the prognosis and accurate stratification of CNS prophylactic therapy. Using PCR technique with specific primers for V, D and J segments of TCRD gene, the pattern of TCRD gene rearrangements in bone marrow lymphoblasts and in cells from cerebrospinal fluid (CSF) have been investigated. The study involved 21 children at the time of diagnosis with B-lineage ALL. In nine of 21 patients incomplete TCRDVD gene rearrangement has been found in CSF cells, which was identical to that observed in bone marrow of the same children. It can be concluded that at least in 43 per cent of all analysed cases, there were signs of CNS involvement in newly diagnosed ALL patients.