Molecular testing are more and more widely used in clinical practice. The premise of clinical application is the verification or validation of the molecular testing procedure. Establishing a verification and validation model suitable for the testing procedures of clinical molecular testing is helpful for the practitioners of clinical molecular testing to carry out the verification and validation work more conveniently and efficiently. It is also helpful for the standardization of testing and quality control significantly. This article will elaborate on the status of verification and validation of molecular testing projects, the requirements of the ISO15189 accreditation system, the establishment of verification validation models and the prospects in the field.

Objective To analyze the structures of atypical class 1 integrons in clinical Proteus isolates. Methods This study included 26 class 1 integron integrase gene-positive clinical Proteus isolates, from which the variable regions of class 1 integrons could not be amplified. Six isolates were chosen from them to amplify the flanking DNA segments of class 1 integron integrase gene using inverse PCR. The se-quences of PCR products were analyzed with BLAST to identify the target homologous sequences as well as their accession numbers in GenBank. Primers for overlap PCR were designed according to the flanking se-quences. Then the 26 clinical Proteus isolates were analyzed with overlap PCR and sequencing analysis. Re-sults The variable regions of class 1 integrons in 25 out of the 26 clinical Proteus isolates were completely or partly identified by using inverse PCR,overlap PCR and sequencing analysis. A gene cassette array of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 was detected in 22 isolates. The variable regions of class 1 integrons in the other three isolates were identified to be estX-psp-aadA2-cmlA1,dfrA14 and IS26,respectively. All of the 25 isolates lacked the 3'conserved segements in class 1 integron. Conclusion Inverse PCR can be used to analyze the structures of atypical class 1 integrons. Gene cassette psp and gene cassette arrays of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 and estX-psp-aadA2-cmlA1 in clinical Proteus isolates are reported for the first time.

Molecular diagnosis plays more and more important role in disease diagnosis, prognosis evaluation and curative effect monitoring.With the support of relevant national policies and the gradual release of accurate medical needs from society, molecular diagnosis relies on its advantages of accuracy, convenience,sensitivity, non-invasiveness and automation to develop rapidly in the direction of precision inspection.The application of molecular diagnostic automation is undoubtedly the catalyst for the development of molecular diagnostics, but its application status is far from satisfactory.It is necessary to analyze the causes,study countermeasures and look forward to the future.

Objective Under the ISO15189 and America Association of Pathologists ( CAP ) laboratory accreditation system, to establish the performance verification standards for detecting hepatitis B virus resistance gene by sanger sequencing.Methods 25 cases of HBV drug resistance outpatients and inpatients were collected from August 2012 to December in Hepatitis Clinic of Huashan Hospital Affiliated to Fudan University.Analytical performance parameters including analytical sensitivity, precision, accuracy, analytical specificity, reference range/reportable range, etc were evaluated.Sequencing quality evaluation parameters included fluorescence signal intensity overall sequencing chromatogram, signal to noise ratio, trace scores and QV value.Results 10%-20% mutation could be detected under wild-type background. The methool had good precision and accuracy.No obvious interference and cross contamination were observed.Conclusions Performance validation of the sequencing should combine with the practical application.Especially in view of the different detection subjects, and appropriately adjusted to meet the clinical needs.Detection of hepatitis B virus resistance gene by the in the test method in this study can be used in clinical detection.

College of American Pathologists (CAP) and International Standard Organization (ISO)15189 accreditation provides requirements to the verification and validation tests for clinical molecular diagnostic assays.The article will introduce the requirements in these aspects including the differences between the two accreditation systems,the differences between the verification and validation tests,the essential factors and standardized framework of the verification and validation tests for clinical molecular diagnostic assays.

Objective To use neutralization confirmatory test to confirm and analyze the results of weak reactive hepatitis B surface antigen(HBsAg) determination by electrochemiluminescence immunoassay(ECLIA).Methods A total of 100 weak reactive HBsAg positive samples(1.00< COI <50.00) were detected by ECLIA,and confirmed by neutralization confirmatory test.The results were analyzed.Results Among the 100 weak reacitve HBsAg positive samples,87 samples(87.0%) were confirmed,10 samples(10%) were negative,and 3 samples(3.0%) were uncertain.According to the receiver operating characteristic(ROC) curve,when the COI was 1.97,the specificity was 100.0%,and the sensitivity was 83.9%.Conclusions The neutralization confirmatory test can be used as the further means to confirm the samples with weak reactive HBsAg.ECLIA shows high sensitivity and specificity on weak reactive HBsAg positive samples.When COI>2.00,generally it is unnecessary to do the neutralization confirmatory test and could exclude the possibility of false-positivity.

>随着定量PCR在实验诊断领域的广泛应用,许多PCR实验室同时使用两台以上不同的荧光定量PCR仪参与日常检验报告的发送。但是,由于不同的检测系统间存在着差异,导致在结果判读时产生疑问:不同仪器系统检测的结果之间究竟存在多大的差异?是否在可接受范围之内?是否需要对结果进行修正?然而,目前国内在评估定量PCR检测的

Objective To detect quantitatively AKAP12 methylation and evaluate its clinical significance in peripheral blood in colorectal cancer. Methods MS-HRM technology was used to detect quantitatively AKAP12 methylation in peripheral blood from 80 colorectal cancer patients and 20 healthy volunteers. They also validated the reproducibility and compared with MSP. Results Thirty-eight of the 80 colorectal cancer samples (47. 5% ) were found to be methylated at the AKAP12 promoter region by MS-HRM (the methylation levels of 24 cancer samples ranged between 1 % and 20% , the methylation levels of 12 cancer samples ranged between 20% and 60% , the methylation levels of 2 cancer samples ranged between 60% and 100% ). The methylation levels of 2 health samples were less than 10% . They also compared the results generated by MS-HRM with a traditional MSP assay. The AKAP12 MS-HRM assay was able to reproducibly detect 1% AKAP12 methylated DNA, whereas the MSP method was unable to detect less than 10% methylation. No significant correlation was observed between the AKAP12 methylation levels and patients' age and gender. However, AKAP12 methylation was significantly higher in DNA from colorectal cancer patients with high Dukes stage and differentiation (x2 =5. 93 or 8. 41, P = 0.01). Conclusions The authors demonstrate here for the first time, the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods have many promising applications in the detection of colorectal cancer.

：Objective To investigatetranscriptional expression and promoter CpG methylation status of A-kinase anchoringprotein 12 (AKAP12) gene and analyze their correlation with clinical pathological stage inbladder transitional cell carcinoma. Methods AKAP12 mRNA expression level and promoter CpGmetbylation status was measured by fluorescent quantitative RT-PCR (FQ-RT-PCR) andmethylation specific PCR (MSP) in 30 bladder transitional cell carcinoma and adjacentnormal tissues. The products of PCR were cloned and bisulfite sequenced. Results DecreasedAKAP12 mRNA expression was demonstrated in 22 carcinomas (73. 3% ) and was significantlyassociated with turnout grade (P =0. 02).The frequency of promoter methylation of AKAP12gene was 53. 3 % (16/30) and correlated with the tumor stage(r =0.52,Pn =0.03)and grade(r=0.61,Pn =0.01). Conclusion Aberrant promoter methylation of AKAP12 can result in the lossof gene expression and may association with bladder transitional cell carcinoma.

Objective:Surface enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) is a no- vel method for biomarker discovery that can provide a rapid protein expression profile.The current study was undertaken to in- vestigate urinary protein profile of isolated impaired fasting glucose patients with normal albuminuria.Methods:Urine of 20 NGT and 22 IFG without elevated ACR were analyzed using surface enhanced laser desorption/ionization time of-flight mass spectrometry (SELDI-TOF-MS) and potential differences in protein or polypeptide profile were identified by Biomarker Wizard Software.Results:Protein profile of urine showed the best peak spectrum on IMAC-Cu2+arrays by sensitivity of 9 and laser intensity of 230.The intensities of protein/polypeptide peaks appeared upregulated in IFG group at M/Z2200、2353、2443、2579、2755、2797、2910、3003、3097、3606,3645、4029 and 5511,but downregulated at M/Z5083.Conclusion:SELDI is powerful and high throughput in comparative proteomic analysis.Small molecular proteins and/or polypeptides excretion may abnormal in IFG with normal urinary albumin.

Purpose To establish a type 2 diabetic retinopathy(DR)specific serum protein fingerprint profile. Methods Sera of 16 healthy volunteers,27 type 2 diabetic patients without DR and 21 type 2 diabetic retinopathy patients were analyzed by using surface enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) and Biomarker Wizard Software to identify potential differences in protein or polypeptide profile. Results Protein profile of serum showed the best peak spectrum on H4 arrays by sensitivity of 9 and laser intensity of 210.Totally 31 mass peaks in the range from 2 000 to 20 000 were detected.The intensities of 5 peaks at m/z 7 440,7 639,7 839,8 147 and 8 925 appeared differentially among three groups(P0.05). Conclusions Although further characterization of differentially detected protein peaks is important and needed,our current work clearly demonstrates that the SELDI is effective in searching biomarkers of DR in serum protein profile.

Objective To investigate the expression of pigment epitheliumderived factor(PEDF) mRNA and its clinical significance in bladder cancer. Methods mRNA of PEDF in 30 bladder tumors and 10 normal tissues were detected by polymerase chain reaction(PCR).The microvessel density was accessed by immunostaining of CD34. Results PEDF was under regulated in 23 of 30 carcinomas(76.6%) and 1 of 10 normal tissues(10.0%).No significant relationship was revealed between PEDF expression and age,tumor size,tumor amount and tumor grade.The loss of PEDF correlated with the malignant stage of tumor and microvessel density. Conclusions This is the first report on PEDF expression in bladder cancer.The loss of this gene might be related with progress of bladder cancer.

随着人类基因组计划的成功实施以及基因组草图的初步完成,人们已经知道了人类基因约有32 000多个.这些信息给我们研究生命活动的奥秘,揭示疾病作用的规律提供了有力的线索.但是如何充分利用基因组测序研究的成果,对生命进行系统、全面的认识,成为一大课题,于是在全球范围内又迅速开展了以功能研究为主的后基因组学研究。

基因研究是20世纪生命科学研究的主要领域.20世纪50年代前,以遗传学为代表,通过对基因分离、连锁等研究,最后提出了DNA双螺旋结构.20世纪中末期,以分子生物学为代表,通过对基因复制、转录、翻译及遗传密码的分析与破译,最终完善了中心法则.随着人类基因组计划(HGP)规模空前的推进和初步完成,基因研究已近顶峰[1],而进入了后基因组时代。

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis and an inducer of neural differentiation. We previously reported the loss of PEDF expression in glioma progression. In this study, we investigated whether PEDF overexpression could suppress glioma growth and invasion. Glioma cell line U251 was stably transfected with a full-length human PEDF expression vector. The expression and release of various cytokines and angiogenic factors into the medium were analyzed by real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. Apoptosis was checked by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Growth inhibition was evaluated by using the in vitro Matrigel invasion. Tumorigenicity was examined in vivo by subcutaneous xenotransplantation into severe combined immunodeficient mice. In U251 cells overexpressing PEDF, thrombospondin-1 protein was upregulated (5.3-fold more), but the production of vascular endothelial growth factor (VEGF) (1.8-fold less) and basic fibroblast growth factor (2.5-fold less) was lower than in cells transfected with the vector only. PEDF also downregulated the production of matrix metalloproteinase-9. Conditioned medium collected from the PEDF-transfected U251 cells showed a significant reduction of VEGF expression. In vitro invasiveness was reduced by approximately 40%. PEDF expression prevented the growth of transfected cells and caused a significant increase in the percentage of cells undergoing apoptosis (50.4% in PEDF-transfected cells). Furthermore, the size of xenotransplants was significantly smaller. In conclusion, PEDF overexpression decreased malignancy, and this might be attributed to the promotion of apoptosis and the regulation of expression of angiogenic effectors. Thus, treatment with PEDF may be useful in patients with malignant gliomas. However, the mechanism of apoptosis induction needs to be investigated.

Objective: To investigate the clinical value of free gastric canncer calls detected in the peritoneal cavity washes by CEA RT-PCR. Methods: A hundered milliliter of peritoneal lavage was obtained from each of 36 patients with advanced gastric cancer and 6 patients with cholecystolithiasis at the beginning operation. A half of the 100 ml peritoneal washes was used for RT-PCR detection while the other part was used for cytopatholgical examination.Gastric cancer cell line MKN-45 was served as positive control. Results: MKN-45 and peritoneal washes of 14 gastric cancer patients (14/36,38.9%),including all 6 patients with positive cytology results (6/36,16.6%)were proved positive for CEA. None of 6 cholecystolithiaasis patients was positive. Moreover, a close association with the depth of cancer invation,lymph node metastasis and tumor differentiation was established. There is one patient recurred intraperitoneally 4 months after curative surgery with positive RT-PCR and negative cytologic results and no patient recurred with negative RT-PCR up to the present. Conclusions: Detection of free cancer cells in peritoneal washes is more reliable by RT-PCR compared with cytology examination, and is promising as a valuble method for prediction of peritoneal recurrence in gastric cancer.

Objective To investigate the expression level of lipocalin-type prostaglandin D synthase (L-PGDS) mRNA and its protein in glioma.Methods The L-PGDS mRNA was determined by real-time quantitative RT-PCR in 23 glioma and 5 healthy brain specimens. L-PGDS protein was detected by Western blot in cerebrospinal fluid (CSF) and brain tissue specimens.Results In 23 glioma specimens L-PGDS mRNA expression ranges from 1.5×10 3～7.0×10 4 copy/μg RNA, mean value is 1.6×10 4 copy/μg RNA. In 5 normal brain specimens, the mean value is 8.1×10 5 copy/μg RNA, statistic analysis indicates a distinct discrepancy ( P0.05), and L-PGDS mRNA expression level correlates with the histological grade of malignancy. For glioma cell line U-251 the expression level of L-PGDS mRNA is 4.7×10 3 copy/μg RNA. Western blot indicates that L-PGDS protein expression level is 0.47±0.12 in glioma and 0.92±0.26 in healthy people, statistic analysis indicates a distinct discrepancy ( P0.01).Conclusions It is found that the expression level of L-PGDS mRNA and its protein decrease in glioma. This implies that L-PGDS has a potential value for diagnosis and therapy of glioma.

Objective To evaluate the role of bacteria in cholesterol pathogenesis. Methods A culture dependent, half quantitative molecular genetic approach was used to detect bacteria from gallstones, bile and gallbladder mucosa in 38 cholesterol gallstone patients and 17 healthy individuals. Immunoglobulin concentrations of bile and gallstone mucosa were also measured in parallel. Results The positive rate of bacteria DNA in bile in gallstone patients and controls was 77% and 67%, with concentrations of 3 23 cfu (log value) and 2 43 cfu, respectively. The positive rate of bacteria DNA in mucosa was 64% and 69%, with concentrations of 3 28 cfu and 3 67 cfu, respectively. No significant differences were found between the 2 groups. No positive correlation was found between immunoglobulin and bacteria concentration. The positive rate of bacteria in core and peripheral part of gallstones was 79% and 82%, with bacteria concentrations of 3 19 cfu and 3 26 cfu, respectively. Bacteria DNA was positive in all the 3 cases of pure cholesterol gallstone. No positive correlation existed between the positive rate of bacteria and the percentage of cholesterol in the stones. There was a sharp distinction for mucosa IgA and IgG concentrations between mucosa bacteria positive and negative ones in gallstone patients. The bile bacteria concentration significantly fell after accepting a dosage of antibiotics right before the operation, while no change on mucosa was observed. Conclusions There is a similar biliary infection rate between gallstone patients and the controls. Finding of bacteria in the biliary tract does not mean the tendency of cholesterol gallstone formation. However, bacteria on the mucosa might indirectly participate in formation of cholesterol gallstone.