Clinical application of multiplex nucleic acid detection technologies: advancements and future / 中华检验医学杂志

With the advantage of being capable of detecting multiple targets at the same time, high throughput and cost-effective, multiplex nucleic acid detection technologies meet the need of large-scale nucleic acid screening and quantification. Multiplex polymerase chain reaction has been applied to detect pathogen, methylated DNA, mutated gene, and single nucleotide polymorphism typing. Isothermal amplification technologies, such as loop-mediated isothermal amplification and recombinase polymerase amplification are promising in the field of point-of-care testing. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-based multiplex nucleic acid detection technologies have become a hotspot due to their high sensitivity and specificity. Metagenomics sequencing plays a leading role in the detection of emerging pathogens and their gene mutation monitoring as well as tumor research. In this review, the advancements and future of multiplex acid detection technologies in clinical application are discussed.

The role of nuclear receptor transcription factor NR2F6 in tumor / 生物工程学报

Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is a member of orphan nuclear receptors, which is expressed in major tissues and organs of the human body, and plays an important role in the regulation of various biological functions and gene expressions. Recent studies have shown that the expression of NR2F6 was up-regulated in a variety of malignant tumors and showed significant correlations with cancer progression. These findings triggered the widespread interest in understanding the relationship between NR2F6 and cancer development and progression. In addition, the latest studies have underscored that NR2F6 was involved in enhancing antitumor immune responses that could serve as a potential target for immune regulation. This review summarizes the biological functions of NR2F6 and its role in tumors, with the aim to provide new insights into effective cancer therapies.

High expression of ZNF652 promotes carcinogenesis and progression of breast cancer / 南方医科大学学报

OBJECTIVE@#To investigate the expression of ZNF652 in breast cancer tissues and cells and explore its role in breast cancer cell proliferation, invasion and migration.@*METHODS@#We exploited the data from the TCGA database to analyze the differential expression of ZNF652 in breast cancer tissues and adjacent tissues and the correlations of ZNF652 expression with the clinicopathological characteristics of breast cancer patients including molecular subtypes, pathological types, TNM stages and clinical stages. RT-qPCR and Western blotting were used to detect the expression of ZNF652 in 5 breast cancer cell lines including MCF-7, MDA-MB-231, SK-BR-3, UACC-812 and BT-474. Using a lentivirus system and siRNA technique, we assessed the effects of ZNF652 over-expression and knockdown on proliferation, colony forming ability, migration and invasion of breast cancer cells with CCK-8 assay, clonogenic assay, Transwell assay and wound healing assay. The subcellular localization of ZNF652 in 293T cells was determined using immunofluorescence assay.@*RESULTS@#ZNF652 was significantly up-regulated in breast cancer tissues (@*CONCLUSIONS@#ZNF652 is highly expressed in breast cancer tissues and cells to promote the development and progression of breast cancer and may serve as a potential molecular target for diagnosis and treatment of the malignancy.

Application of laboratory diagnostic technologies for SARS-CoV-2: current progress and prospect / 南方医科大学学报

Since the outbreak of COVID-19 pandemic, the detection capability has been improving and the detection techniques have been evolving with innovations. qRT- PCR and mNGS, which represent the current mainstay diagnostic technologies, play key roles in disease diagnosis and monitoring of virus variation. The detection technologies based on serum and plasma IgM and IgG antibodies are important for auxiliary diagnosis. RT-LAMP is highly specific for a diagnostic purpose. Digital PCR could quantitatively detect nucleic acid and SHERLOCK has a higher sensitivity. These techniques all have great potential for future development and application for pathogen detection. In this review the authors summarize the basic rationales, technical characteristics and the current application of the SARS-CoV-2 detection techniques.

Prevention and control of SARS-CoV-2 infection in clinical laboratories: implementation of contingency plan and postpandemic response strategies / 南方医科大学学报

The outbreak of COVID-19 has currently been under control in China, but now the disease has rapidly evolved into a global pandemic. We formulated a prevention and control plan for clinical laboratories responsible for detection of the novel coronavirus infection. We analyzed the implementation of this plan and the problems arising from its clinical practice. We found that the layout of most clinical laboratories (including gene amplification laboratories for clinical samples) was inadequate in response to a major outbreak and did not meet the requirements for biosafety protection and etiology and serology testing; and laboratory staff showed insufficiencies in their awareness regarding biosafety protection; the functions and status of the laboratory in the fever clinic need to be enhanced to increase its detection capacity; the high density of military personnel, the low level of automation of clinical laboratory equipment, and the lack of biosafety cabinets and personal protective equipment all limit the performance of diverse military operations and major overseas missions. In view of these problems, we propose the following strategies and recommendations: the clinical laboratory needs to standardize the design and staff management according to the standards of P2 laboratory; the detection capacity and staffing of fever clinic laboratory in hospitals need to be strengthened, and a separate clinical gene amplification laboratory can be optimal; for those clinical gene amplification laboratories that fail to meet these standards, reconstruction and upgrade should be made according to the requirements of biosafety protection; for the clinical laboratory in the military medical system, in addition to enforcement of biological safety protection of the staff, sufficient supply of medical materials and biological safety equipment should be ensured and biological safety cabinets should be routinely equipped if possible.

Expression of CXCR family proteins in different subtypes of breast cancer and their clinical prognosis / 实用肿瘤学杂志

Objective The aims of this study were to determine the expression of CXCR family proteins in various subtypes and adjacent tissues of breast cancer and its relationship with prognosis,and to provide a reference for clinical diagnosis,treatment and prognosis of breast cancer. Methods The mRNA expressive profiles of CXCR family proteins in paracancerous tissues and different subtypes of breast cancer tissues were obtained from the TCGA(The Cancer Genome Atlas)database. The prognostic survival analysis of each differentially expressed protein was obtained using the PRECOG website. Results Except for CXCR1,the expression of CX-CR family proteins in breast cancer tissues and adjacent tissues was statistically different( P<0. 05). CXCR2P1,CXCR3,CXCR4, CXCR5 and CXCR6 were highly expressed in breast cancer tissues,CXCR2 and CXCR7 were lowly expressed in breast cancer tis-sues. The expressive levels of CXCR3,CXCR4 and CXCR7 were associated with the prognosis of patients. Conclusion The expres-sions of CXCR3,CXCR4 and CXCR7 in breast cancer tissues and adjacent tissues is significantly different,and its expression is related to the prognosis of breast cancer patients,which may be a potential target for molecular diagnosis or targeted therapy of breast cancer.

Expression of serum miR-638 in breast cancer and its clinical value / 临床检验杂志

Objective@#To investigate the expression levels of serum miR-638 in the patients with breast cancer and its clinical value. @*Methods@#One hundred and fifty-two patients with breast cancer were selected as the disease group, and 102 healthy persons as the control group. The expression levels of miR-638 in their serum samples were detected by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the expression levels of serum miR-638 in the patients with different pathological stages, before and after operation, and before and after chemotherapy were compared. The diagnosis efficacy of serum miR-638 alone and in combination with CEA, CA125, CA15-3 for breast cancer was analyzed by the ROC curve and Z test. @*Results@#The expression levels of serum miR-638 in the breast cancer group (3.6 [1.3～10.5]) were significantly lower than that in the control group (79.0 [52.5～120.8],P＜0.01). The linear regression analysis showed that chemotherapy and pathological staging were the main factors influencing the expression levels of serum miR-638. The area under the ROC curve (AUC ROC ) of serum miR-638 in the diagnosis of breast cancer was 0.954. When the cut-off value of serum miR-638 was 27.47, its sensitivity and specificity for the diagnosis of breast cancer were 94% and 86.2%, respectively. The area under the ROC curve (AUC ROC ) of serum miR-638 combined with CEA, CA125 and CA15-3 in the diagnosis of breast cancer was 0.978 8. When the combined cut-off value was 0.29, their sensitivity and specificity for the diagnosis of breast cancer were 95.4% and 89.5%, respectively. There was no significant difference in the AUC ROC between miR-638 alone and combined screening (Z=1.68,P=0.091). @*Conclusion@#Serum miR-638 may be a potential molecular marker for the screening of breast cancer.

Structure and function of B-cell linker and its role in the development of B cell-related diseases / 南方医科大学学报

B cell linker (BLNK) is a key linker protein of B cell receptor (BCR) signaling pathway. BLNK participates in the regulation of PLC-γactivity and the activation of Ras pathway through its typical structure and interaction network with other proteins, and is thus widely involved in the regulation of B cell proliferation, differentiation, apoptosis and signal transduction. Furthermore, it is closely related to anaphylactic diseases, multiple sclerosis, chromosomal aneuploidy, aneuglobulinemia, B lymphocytic leukemia and lymphoma. Herein we review the structure and biological function of BLNK and its role in B cell-related diseases. BLNK can cooperate with a series of effective proteins to activate BCR signaling pathway, thereby regulating the development, maturation and function of B cells. The functional mutation of BLNK can destroy the homeostasis of B cells and affect the development and maturation of B cells, which leads to the occurrence of B cell related diseases. A comprehensive understanding of the biological functions of BLNK not only provides insights into the pathogenesis of B cell-related diseases, but also inspires new ideas and helps to find breakthroughs for the treatment of these diseases with BLNK as the therapeutic target.

Research of peripheral blood non-coding RNA in diagnosis of breast cancer / 中华检验医学杂志

A large amount of non-coding RNA are found existing in eukaryotic transcriptome, which play an important role in regulating the expression of genes as well as participating in physiological and pathological process of various cells.In recent years,the research of ncRNA in diagnosis of breast cancer is extremely hot. In this review, the research of three types of peripheral blood ncRNAs as breast cancer diagnostic biomarkers were reviewed, including microRNA(miRNA), long non-coding RNA(lncRNA), and circular RNA(circRNA).Also,the prospect of ncRNA in clinical research and translational medicine of breast cancer was mentioned, in order to provide a new idea for breast cancer with the discovery of diagnostic markers in early detection and the monitoring of diagnosis and treatment process.

Differences in circRNA expression profiles between HER-2-positive breast cancer cells and normal mammary epithelial cells / 中华肿瘤杂志

Objective@#To investigate the differential expression profiles of circular RNA (circRNA) in human epidermal growth factor receptor 2 (HER-2) positive breast cancer cells and normal mammary epithelial cells, and to develop novel diagnostic and therapeutic markers for HER-2 positive breast cancer.@*Methods@#Total RNA were extracted from HER-2 positive breast cancer cell SK-BR-3 and normal mammary epithelial cell MCF10A. RNA quality was detected using NanoDrop ND-1000. Rnase R was applied to remove linear RNA and enrich circRNAs. After amplification and reverse transcription into fluorescent complementary RNA (cRNA) using random primer, the labeled cRNAs were hybridized onto the Arraystar Human circRNA Arrays. The raw data were extracted and the acquired array images were subjected to quantile normalization followed by heat map and volcano plot analysis. The expression of circRNAs with large fold change was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in the differentially expressed circRNAs and circRNA-microRNA (miRNA) network was constructed.@*Results@#The total RNA extracted from SK-BR-3 and MCF10A had high integrity and quality. The expression profiles of circRNA in SK-BR-3 and MCF10A cells were significantly different shown by fluorescent expression signals. Compared with MCF10A cells, there were 6 584 up-regulated circRNAs and 6254 down-regulated circRNAs in SK-BR-3 cells. There were 348 circRNAs with |log2FC|≥2, of which 153 were up-regulated and 195 were down-regulated. Moreover, 8 circRNAs with |log2FC|>5. Among them, 5 were up-regulated in SK-BR-3 cells, including hsa_circRNA_074595 (|log2FC|=7.84), hsa_circRNA_074598 (|log2FC|=6.50), hsa_circRNA_085362 (|log2FC|=5.86), hsa_circRNA_101379 (|log2FC|=5.71) and hsa_circRNA_406683 (|log2FC|=5.34); as well as 3 were down-regulated, including hsa_circRNA_021714 (|log2FC|=5.46), hsa_circRNA_100777 (|log2FC|=5.40), and hsa_circRNA_100796 (|log2FC|=5.03). The expression levels of hsa_circRNA_074595, hsa_circRNA_074598 and hsa_circRNA_100777 were further validated by RT-qPCR in consistent with the results of microarray. GO analysis showed that differentially expressed circRNAs were significantly enriched in the biological process of heart development (P<0.001), cellular component in the cell adhesion-related components (P<0.001), molecular function in protein serine/threonine kinase activity (P<0.001). KEGG analysis revealed that differentially expressed circRNAs were significantly enriched in the PI3K-Akt signaling pathway.@*Conclusions@#The expression profile of circRNA in HER-2 positive breast cancer cells is significantly different from that in normal mammary epithelial cells. The differentially expressed circRNAs may be served as potential diagnostic or therapeutic targets for HER-2 positive breast cancer.

Research of peripheral blood non-coding RNA in diagnosis of breast cancer / 中华检验医学杂志

A large amount of non-coding RNA are found existing in eukaryotic transcriptome, which play an important role in regulating the expression of genes as well as participating in physiological and pathological process of various cells.In recent years,the research of ncRNA in diagnosis of breast cancer is extremely hot. In this review, the research of three types of peripheral blood ncRNAs as breast cancer diagnostic biomarkers were reviewed, including microRNA(miRNA), long non-coding RNA(lncRNA), and circular RNA(circRNA).Also,the prospect of ncRNA in clinical research and translational medicine of breast cancer was mentioned, in order to provide a new idea for breast cancer with the discovery of diagnostic markers in early detection and the monitoring of diagnosis and treatment process.

Expression and purification of GRIK3 intracellular region protein in prokaryotic cell system / 实用医学杂志

Objective To express and purify GRIK3 intracellular region protein in prokaryotic cell system. Methods The histamine(His)tagged GRIK3 intracellular region recombinant plasmid pCzn1-GRIK3-in-tra was constructed and transformed into Escherichia coli(E.coli). The His-GRIK3-intra recombinant protein was expressed after the induction with IPTG. The target protein was purified by Ni-NTA affinity chromatography column. Results The prokaryotic expression plasmid pCzn1-GRIK3- intra was successfully constructed and the GRIK3 intracellular region protein in E.coli were efficiently expressed after the induction with IPTG. The purified target pro-tein was successfully obtained by Ni-NTA affinity chromatography column. Conclusion The successful construc-tion of prokaryotic expression plasmid expressing GRIK3 intracellular region gene and preparation of high purity GRIK3 intracellular region protein have paved the way for further exploration of the intracellular signal transduction mechanism of membrane protein GRIK3.

Clinical application of serum miR-122-5p and miR-486-5p in the diagnosis of hepatocellular carcinoma / 中华检验医学杂志

Objective Explore the relative expression of miR-122-5p and miR-486-5p in the serum of Hepatocellular carcinoma ( HCC) patients and its clinical value .Methods Case-control study was used in this research.From June of 2016 to March of 2017,60 HCC patients who were hospitalized in Guangzhou General Hospital were selected as HCC group .It also selected 20 hepatitis patients ( hepatitis group ) , 20 cirrhosis patients ( cirrhosis group ) , 20 breast cancer patients ( breast cancer group ) , 20 gastric cancer patients(gastric cancer group)and 20 healthy controls (normal control group) for comparison.The relative expression of miR-122-5p and miR-486-5p was detected by real-time fluorescent quantitative PCR.The specificity and sensitivity of miRNAs for the diagnosis of HCC were analyzed by receiver operating characteristic ( ROC) , and the results were compared with the tumor marker AFP .The effect of miRNA on the diagnosis of hepatocellular carcinoma was evaluated by the area under the ROC curve , which was used to detect the diagnostic efficiency of liver cancer .SPSS22.0 statistical software was used for statistical analysis . The rank sum test was applied in the group comparison .Results Serum levels of miR-122-5p in HCC group, hepatitis group, cirrhosis group, breast cancer group, gastric cancer group and control group were 0.14(0.05-0.51),0.45(0.32-0.58),0.53(0.34-0.67),0.14(0.07-0.28),0.29(0.13-0.36) and 0.73 (0.63-0.95),respectively, and the miR-486-5p were 0.50(0.23-0.77),0.62(0.48-0.82),0.65(0.54-0.85),0.23(0.08-0.40),0.29(0.15-0.45)and 0.76(0.69-1.23).The serum levels of miR-122-5p in hepatitis group , cirrhosis group , HCC group were significantly lower in healthy control group , significance was found (U was 315.37,393.46,429.08, all P<0.01), and the serum levels of miR-486-5p in hepatitis group, cirrhosis group, HCC group were lower in healthy control group , significance was found ( U was 103.67,156.18,207.35, all P<0.05).When using one serum marker to diagnosis HCC , AFP had the highest sensitivity ( 73.7％) and miR-122-5p had the highest specificity ( 95％) .While combined two serum markers, AFP +miR-122-5p had the highest sensitivity and specificity (93％),and miR-122-5p +miR-486-5p had the highest specificity (70％), compared AFP +miR-122-5p to AFP, AUC difference was statistically significant(Z=3.02,P<0.01), while there was no significant difference in AUC with AFP +miR-486-5p, miR-122-5p +miR-486-5p to AFP(Z=1.57,1.39,all P>0.05).The sensitivity and specificity of the three markers were 96.5％and 55％respectively , and the area under the ROC curve was 0.891 (95％CI:0.818-0.964).The combination miR-122-5p, miR-486-5p and AFP were higher than the single test, compared with AFP, miR-122-5p, miR-486-5p, the AUC differences was statistically significant (Z=3.26, 3.72, 4.25, all P<0.01).Conclusion Serum miR-122-5p and miR-486-5p could be used as biological markers for the diagnosis of HCC .

Differences in expression profiles of circular RNA between luminal breast cancer cells and normal breast cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differences in the expression profiles of circular RNA (circRNA) between luminal breast cancer cells and normal breast cells.</p><p><b>METHODS</b>Total RNA extracted from luminal breast cancer cells MCF7 and normal breast cells MCF10A was digested with Rnase R to remove linear RNAs and enrich circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNAs using a random priming method, and were hybridized onto the circRNA hybridization array. The circRNA expression profiles of MCF7 and MCF10A cells were analyzed using Agilent Feature Extraction software. Quantile normalization and subsequent data processing were performed, and volcano plot filtering and hierarchical clustering were utilized to analyze the circRNA expression patterns. The expressions of 3 circRNAs with significant log fold changes were validated using qPCR.</p><p><b>RESULTS</b>The hybridization array data revealed significant differences in the circRNA expression profiles between MCF7 and MCF10A cells. Compared with those of MCF10A cells, the 12910 circRNAs expressed in MCF7 cells showed 5964 up-regulated, 81 consistently regulated, and 6865 down-regulated circRNAs; 343 circRNAs showed a log fold change by more than 2 folds, among which 213 circRNAs were up-regulated and 130 were down-regulated. Nine circRNAs showed differential expressions by more than 2 folds, including 8 up-regulated ones, namely hsa_circRNA_061260 (6.02 folds), hsa_circRNA_103933 (5.96 folds), hsa_circRNA_005239 (5.84 folds), hsa_circRNA_100689 (5.69 folds), hsa_circRNA_004087 (5.60 folds), hsa_circRNA_104420 (5.25 folds), hsa_circRNA_104421 (5.13 folds) and hsa_circRNA_101222 (5.03 folds); only one circRNA was down-regulated, namely hsa_circRNA_104864 (5.09 folds). The expressions of hsa_circRNA_100689, hsa_circRNA_005239 and hsa_circRNA_104864 were further validated by qPCR, which yielded consistent results with the microarray data.</p><p><b>CONCLUSIONS</b>The circRNA expression profiles differ significantly between luminal breast cancer cells and normal breast cells. These differentially expressed circRNAs may serve as potential novel targets for the diagnosis of luminal breast cancer.</p>

Mutations in C region of hepatitis B virus may contribute to occult hepatitis B virus infection / 实用医学杂志

Objective To investigate the role of mutations in C region that may contribute to occult hepatitis B virus infection.Methods C genes were amplified from two OBI blood donor samples respectively.Plasmids with mutations in C region of hepatitis B virus were constructed by overlapping PCR.HBsAg and HBeAg in Huh7 cells and in the serum of Balb/c mice were detected by CMLA.HBcAg in liver tissue was detected by immunohistochemistry,while HBV-RNA was tested by RT-PCR.Results Mutations in C region significantly reduced the expression level of HBeAg and HBcAg,but had no significant effect on HBsAg and HBV-RNA.Conclusion The mutations in C region affect the expression level of HBeAg and HBcAg,which may play an important role in the occurrence of OBI.

Construction of human kinase knock-out library by using CRISPR/Cas9 technique / 实用医学杂志

Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.

The clinical application and research progression of some canonical tumor biomarkers / 中华检验医学杂志

Early detection and diagnosis are the key steps in curing tumors .With the development of molecular biology technology , tumor biomarkers detection plays an increasingly important role in many aspects, such as looking for primary tumors , screening tumor susceptible persons and estimating the progression of various tumors .The number of frequently used tumor biomarkers in early detection , guiding treatment and estimating prognosis is limited .All these tumor biomarkers show their own advantages and certain limitations in clinical application .The tumor markers used in targeting cell proliferation are hopeful in promoting the rate of tumor detection .

Effect of clopidogrel on neck blood vessel stenosis and plasma fibrinogen in patients with SIP / 中国生化药物杂志

Objective To study effect of clopidogrel on neck blood vessel stenosis and plasma fibrinogen ( FIB ) in patients with stroke in progression(SIP).Methods 80 cases with SIP were selected from March 2014 to March 2015, they were divided into study group and control group according to the random number table method, 40 cases in each group, the control group was received conventional treatment, the study group was given clopidogrel on the basis of conventional treatment, evaluation of neurological function and living ability of patients with Stroke Scale (NIHSS) and daily living ability score ( ADL) , carotid artery stenosis were measured by color Doppler, the levels of FIB and C reactive protein ( hs-CRP) in two groups were also measured, adverse reactions in the two groups were compared.Results Carotid artery, internal carotid artery, external carotid artery diameter before treatment between two groups were no statistical significance,which all increased after treatment, and the study group was wider than the control group, the difference was statistically significant (P <0.05);NIHSS score and ADL score before the treatment between two groups was no statistical significance.NIHSS score and ADL score after treatment between two groups were significant improved, and the study group was better than the control group, the difference was statistically significant (P<0.05); FIB and hs-CRP before the treatment between two groups was no statistical significance, FIB and hs-CRP levels after treatment between two groups were significantly reduced, the study group was significantly lower than the control group, the difference was statistically significant (P<0.05); the adverse reactions of the two groups were not statistically significant.Conclusion Clopidogrel can significantly improve the neurological function, improve the degree of cervical vascular stenosis, reduce the level of FIB for SIP patients in recovery period.

Preparation of a monoclonal antibody against fusion protein preM/EIII of WNV / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a monoclonal antibody (mAb) against the fusion protein preM/EIII of West Nile virus (WNV) for clinical detection of WNV.</p><p><b>METHODS</b>Sp2/0 cells were fused with the spleen cells of BALB/c mice immunized with the recombinant fusion protein preM/EIII expressed in E. coil to obtain the hybridoma cell line that secreted preM/EIII mAb. The hybridoma cells were injected into the peritoneal cavity of BALB/c mice and the ascites was collected and purified. The specificity and titer of the obtained mAb were determined using ELISA and Western blotting.</p><p><b>RESULTS</b>One hybridoma cell line secreting preM/EIII mAb, named ME1, was obtained. The titer of the purified mAb was 10(-6). Identified as a mAb of the Ig subclass IgG1, ME1 was capable of specific reactions with preM/EIII protein and WNV without cross-reactions with other viruses such as JEV, SLEV, YFV and DENV. The accuracy of clinical testing of MNV with ME1 was 97.78%.</p><p><b>CONCLUSION</b>The mAb against preM/EIII obtained have a high specificity and accuracy in clinical testing of MNV and can be used in clinical diagnosis of MNV infection.</p>

Association between SNP rs10569304 on the second expressed region of hole gene and the congenital heart disease / 华中科技大学学报（医学）（英德文版）

The correlation of single nucleotide polymorphism (SNP) rs10569304 on the second expressed region of hole gene and congenital heart disease (CHD) of human being, and the effect of hole gene on CHD were investigated. 179 patients with CHD as CHD group and 183 healthy people as control group were selected in the case-control study. DNA was abstracted from the peripheral blood by phenol-chloroform method. Primer was designed for the flanking sequence of SNP rs10569304 on the second expressed region of hole gene. The genotype was identified by PCR degenerative acrylamide electrophoresis with amplification products. Then the three amplification products received sequencing. By chi-square test, the genotype frequency and allele frequency in CHD group and control group were analyzed. There was insertion-deletion (GCC/-) of SNP rs10569304 which corresponded to alleles of A and B in Southern Chinese people. The genotype frequency and allele frequency in control group and CHD group were met the Hardy-Weinberg equilibrium. By chi-square test, in control group and CHD group, the genotype frequency of AA (insertion homozygous), AB (insertion-deletion heterozygous) and BB (deletion homozygous) was 21.31%, 54.09%, 24.59% and 16.75%, 46.36%, 36.87%, respectively. The distributional difference of genotype frequency had statistical significance (chi (2)=6.51, P<0.05); The allele frequency of A and B was 48.36% and 51.64% in control group, 39.94% and 60.06% in CHD group, respectively. The distributional difference of allele frequency had statistical significance (chi (2)=5.20, P<0.05). Meanwhile, by contrast with the control group, the BB genotype frequency and B allele frequency in CHD group was higher, but the AA and AB frequency was lower. There was higher risk to suffer from CHD involving B allele. BB genotype had 1.907-fold increased risk of developing CHD according to AA genotype (P<0.05). It is concluded that there is insertion-deletion (GCC/-) of SNP rs10569304 in the Southern Chinese people, and the people whose hole gene involving BB genotype have higher risk to suffering from CHD.