探讨绒毛细胞长期培养法在产前诊断及早孕和早中孕期自然流产病因检测中的应用价值

目的探究产前诊断及早孕和早中孕期自然流产病因检测中绒毛细胞长期培养法的应用效果,另分析产前诊断及早孕和早中孕期自然流产时期绒毛细胞长期培养法的检测结果差异。方法共计抽取1058例患者参与本次研究,其中使用B超引导进行产前诊断的患者236例,早孕和早中孕期自然流产患者822例,患者均于2015年12月-2019年11月入我院进行检查治疗,分组对比患者检测结果。结果绒毛细胞长期培养在产前诊断中绒毛培养的成功率与早孕和早中孕期自然流产中的成功率相比较高,早孕和早中孕期自然流产绒毛染色体异常率与产前检查相比明显较高,差异有统计学意义(P<0.05)。两组患者绒毛细胞长期培养法的培养时间分组对比无明显差异,无统计学意义(P>0.05)。结论绒毛细胞长期培养在临床中的应用可以作为产前诊断以及流产诊断的标志,临床诊断中需要确保检验标本的新鲜度以及无菌性,严格按照要求进行绒毛细胞培养,为临床诊断及治疗提供基础。

婴儿肝炎综合征临床病毒病因检测分析报告

婴儿肝炎综合征（HIS）以综合治疗为主，应尽早找出病因，针对病因进行治疗；加强母孕期保健，及早进行相应的干预处理是预防IHS的关键。

Genetic Variation Analysis of 3D Gene and Molecular Detection of Porcine Kobuvirus in 2013-2014

[Objective] This study aimed to investigate the prevalence and variation of porcine kobuvirus （PKV） in suckling piglets in China. [Method] In 2013-2014, 224 feces samples from suckling piglets with diarrhea in 27 pig farms of five provinces in China were collected to detect 3D genes of PKV with RT-PCR method; the sequences and genetic variation of 29 PKV 3D genes were analyzed. [Result] Total positive rate of PKV in feces samples from suckling piglets with diarrhea was 65.18% （146/224）; total positive rate of PKV in pig farms was 85,2% （23/27）; nucleotide sequences and the deduced amino acid sequences of 29 PKV 3D genes shared 87.0%-100% and 92.7%-100% homologies with six PKV-related 3D sequences, respectively. [Conclusion] PKV infection is prevalent in suckling piglets in China; PKV 3D genes exhibit high diversity.

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

AIM： TO determine the genotype distribution of hepatitis B virus （HBV） with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS： An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing. RESULTS： HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for 8.3% （n = 33）, 83.2% （n = 333）, and 8.5% （n = 34）, respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing. CONCLUSION： The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.

Establishment of an ELISA to Detect Kaposi's Sarcoma-associated Herpesvirus Using Recombinant ORF73

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a proportion of cases of multicentric Castleman's disease (MCD). The ORF73 protein was cloned into pQE80L-orf73 and expressed in E.coli and purified. The expressed recombinant ORF73 was identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A protein of about 27 kDa was expressed as expected. Western Blotting showed that the purified recombinant ORF73 reacted with KSHV positive serum. The immunogenicity of the recombinant ORF73 was further analysed by ELISA and the optimal conditions were determined. The ORF73 ELISA was used to compare the KSHV seroprevalence between Hubei and Xinjiang Han people. The Han people in Xinjiang have significantly higher KSHV seroprevalence than their counterparts in Hubei (6.7% vs 2.9%, P = 0.005).

Importance of early diagnosis of pancreaticobiliary maljunction without biliary dilatation

AIM:To clarify the strategy for early diagnosis of pancreaticobiliary maljunction(PBM) without biliary dilatation and to pathologically examine gallbladder before cancer develops.METHODS:The anatomy of the union of the pancreatic and bile ducts was assessed by using endoscopic retrograde cholangiopancreatography(ERCP).Patients with a long common channel in which communication between the pancreatic and bile ducts was maintained even during sphincter contraction were diagnosed as having PBM.Of these,patients in which the maximal diameter of the bile duct was less than 10 mm were diagnosed with PBM without biliary dilatation.The process of diagnosing 54 patients with PBM without biliary dilatation was retrospectively investigated.Histopathological analysis of resected gallbladder specimens from 8 patients with PBM without biliary dilatation or cancer was conducted.RESULTS:Thirty-six PBM patients without biliary dilatation were diagnosed with gallbladder cancer after showing clinical symptoms such as abdominal or back pain(n = 16) or jaundice(n = 12).Radical surgery for gallbladder cancer was only possible in 11 patients(31%) and only 4 patients(11%) survived for 5 years.Eight patients were suspected as having PBM without biliary dilatation from the finding of gallbladder wall thickening on ultrasound and the diagnosis was confirmed by ERCP and/or magnetic resonance cholangiopancreatography(MRCP).The median age of these 8 patients was younger by a decade than PBM patients with gallbladder cancer.All 8 patients underwent prophylactic cholecystectomy and bile duct cancer has not occurred.Wall thickness and mucosal height of the 8 resected gallbladders were significantly greater than controls,and hyperplastic changes,hypertrophic muscular layer,subserosal fibrosis,and adenomyomatosis were detected in 7(88%),5(63%),7(88%) and 5(63%) patients,respectively.Ki-67 labeling index was high and K-ras mutation was detected in 3 of 6 patients.CONCLUSION:To detect PBM without biliary dilatation before onset of gallbladder cancer,we should perform MRCP for individuals showing increased gallbladder wall thickness on ultrasound.

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.

宏基因组二代测序技术辅助诊断内脏利什曼病1例

1例患者因“反复高热、肝脾肿大、伴全血细胞减少待查”收住入院,排除细菌、病毒、真菌感染及血液系统疾病后,采集患者外周血行宏基因组二代测序,结果显示婴儿利什曼原虫感染。rk39快速诊断试剂检测显示抗利什曼原虫抗体阳性,骨髓涂片检出利什曼原虫无鞭毛体,最终确诊为内脏利什曼病。给予葡萄糖酸锑钠、激素进行抗感染、保肝、升白细胞等治疗及个性化护理后,患者康复出院。宏基因组测序技术在不明原因发热患者病因检测中具有重要价值,值得临床推广应用。

The diagnosis and treatment process of one clinical case of juvenile glycogen storage disease in current medical model in china

Glycogen storage disease type Ⅱ, also known as Pompe disease (PD), is a kind of congenital metabolic myopathy, the cause of this disease is the barrier of glycogen disintegration due to the shortage of acid alpha-1,4-glucosidase enzyme. The prevalence of PD ranges between 1:40,000 and 1:300,000 and is dependent on ethnic and geographical factors. The main clinical manifestation of this myopathy is the injury of muscle organization. According to the difference in age and developmental speed, we can divide this disease into three types: the infantile type, the juvenile type and the adult type, and the latter two types are called the late onset type. Because the symptom of the late onset form is not typical, the mutual mixture of the late onset type and some chronic myopathy always takes place, which can even cause evade diagnosis and misdiagnosis, thus it is supposed to be highly focused on. At present, there is a shortage of effective therapeutic methods to cope with this myopathy all over the world. The following part is a report about the clinical data and the rehabilitation intervention of a patient who caught the juvenile type of GSD-Ⅱ, which is combined to home and abroad literatures, the purpose of this report is to help enhance clinical physicians’ cognition of this disease in the future therapy. At the same time, we combine with the current medical mode and system in China to reflect on the prevention and treatment of such diseases.

B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160 （epitopel）, tandem repeat 200-213 （epitope2 （＋2）） and the combination of two epitopes （epitopel-2） was genetically cloned into the prokaryotic expression vector PPRoExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 （＋2） and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 （＋2）, GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

Detection of Hantavirus gene from peripheral blood of patients with HFRS in Heilongjiang province and the epidemiological significance

The aim of this study is to further understand the genotype of Hantavirus （HV） from peripheral blood of patients with hemorrhagic fever with renal syndrome （HFRS） and the epidemiological significance of this disease in Heilongjiang province in recent years. Thirty-one serum samples of clinically diagnosed patients with HFRS were examined by RT-PCR to decide the genetic subtype. On the basis of infection season, the serum samples were divided into two groups： winter （Nov, 2003--Feb, 2004）, spring and summer （April, 200d--Sep, 2004）. Further analysis was performed in combination with clinical symptoms. It was found that among the total 31 samples, 22 were sero-positive. Among 14 serum samples in winter, 8 were sero-positive, of which 5 cases were of type Ⅰ （Hantaan virus, HTNV） and 3 of type Ⅱ （Seoul virus, SEOV）. Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of type Ⅰ and 9 of type Ⅱ . So it concludes that both of the two types of Hantavirus exist in Heilongjiang. The type Ⅰ is the main pathogen of HFRS in winter, and typeⅡ is the main in spring and summer.

Investigation on detection of Haemophilus ducreyi by Polymerase Chain Reaction

Objective:To investigate the application of polymerase chain reaction (PCR) detection of Haemophilus ducreyi in clinical diagnosis of chancroid. Methods: Nucleotide sequences of 16srRNA gene specific for H. dureyi were used to develop primer sets for amplification of two strains. The amplified products were tested via PCR and sequenced by electrophoresis in a 1.5% gel.These products were compared with those of heterogeneous species or related bacteria to test the specificity of the PCR assay. PCR amplification with different concentrations of H.ducreyi was performed to test its sensitivity. Results: PCR amplification of two strains of H. ducreyi produced a single band of expected 438bp length. The sequence was identified with genomic DNA. None of the other 19 reference species amplified under the same conditions gave this result. The highest sensitivity of PCR assay in the present test was 10ng/L. Conclusions: PCR assay for detection of H. ducreyi is a rapid, specific, and sensitive detection method. If laboratory conditions are strictly controlled, PCR assay is a potentially useful laboratory test for H. ducreyi infection diagnosis.

The expression of Twist in salivary adenoid cystic carcinoma and its clinicopathological significance

Objective:The purpose of this study was to investigate the expression of Twist in salivary adenoid cystic carcinoma (SACC) and the relations between Twist expression and the clinicopathological characteristics. Methods: The expression of Twist was examined immunohistochemically in 48 cases of SACC, 18 of pleomorphic adenoma and 10 of normal parotid gland. The relationship between Twist expression in SACC and the clinicopathological factors was analyzed. Results: Twist expression was significantly higher in SACC tissues compared with pleomorphic adenomas and normal parotid gland tissues (P<0.05). Twist expression levels in SACC were significantly associated with histological type, perineural invasion, postoperative recurrence, and distant metastasis (P<0.05), rather than age, gender, tumor site, tumor size, or clinical staging (P>0.05). Conclusion: Twist expression may be associated with tumor cell differentiation, perineural invasion and metastasis of SACC. Twist expression is a potential prognostic factor for disease survival of SACC.

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

A new polymorphism in the GRP78 is not associated with HBV invasion

AIM:To examine the association between -86 bp(T>A) in the glucose-regulated protein 78 gene(GRP78) and hepatitis B virus(HBV) invasion.METHODS:DNA was genotyped for the single-nucleotide polymorphism by polymerase chain reaction followed by sequencing in a sample of 382 unrelated HBV carriers and a total of 350 sex-and age-matched healthy controls.Serological markers for HBV infection were determined by enzyme-linked immunosorbent as-say kits or clinical chemistry testing.RESULTS:The distributions of allelotype and genotype in cases were not significantly different from those in controls.In addition,our fi ndings suggested that neither alanine aminotransferase/hepatitis B e antigen nor HBV-DNA were associated with the allele/genotype variation in HBV infected individuals.CONCLUSION:-86 bp T>A polymorphism in GRP78 gene is not related to the clinical risk and acute exacerbation of HBV invasion.

675例病毒性肝炎病原检测

６７５例病毒性肝炎病原检测皖南医学院弋矶山医院（２４１０１）余家宽为了解病毒性肝炎病原学分布，对６７５例各型肝炎检测了甲乙丙三型肝炎病霉血清标志物现报告如下。１病例来源及方法１．１病例来源均为住院病人，按１９９０年上海第六次全国病毒性肝炎学术会议诊断...

Genotyping and preparation of the recombinant nucleocapsid protein antigen of hantavirus

Objective To identify new recombinant antigens with potential for diagnosis of hemorrhagic fever with renal syndrom (HFRS) and establish reverse transcription-polymerase chain reaction-restriction fragments length polymorphism (RT-PCR-RFLP) for genotyping of hantavirus.Methods One group of primers was used to clone the full-length S genome segment and the partial S genorme segment of the N-terminal. The two cloned genes were both fusionally expressed and nonfusionally expressed in the T7 system. The other group of primers was used to establish a RT-PCR method to detect RNAs in 37 virus isolates, 2 positive standard virus strains of hantavirus and 5 negative controls.The method for typing RFLP was set up by digesting the PCR products of 20 virus isolates with Ras Ⅰ and Hind Ⅲ.Results The non-fusionally expressed products with a working concentration of 1:10 000 by chapping enzyme-linked immunosorbent assay (cELISA), presented good biological activity though yields were lower than that of the fusionally expressed products.The specific component of the hantavirus genome (299 bp or 577 bp) wes seen in all viral samples. The genotyping of hantavirus showed that 9 out of the total were hantann (HTN) viruses, 8 were seol (SEO) viruses and 3 were not determined.Conclusions The good working titrer of expressed recombinant antigen showed that it has the potential to replace the natural antigen for detecting hantavirus antibodies. On comparison with cELISA, the detection rates for these two methods were 100% and 84.6%, and the coincidence rate was 84.6%. The former had a 15.4% higher sensitivity than the latter. The typing efficiency of RT-PCR-RFLP and sero-typing method was 85% (17/20) and 55% (11/20), respectively, showing that the former was 30% higher than the latter, while their results were highly consistant.