Objective To develop a PCR-ELISA assay for the rapid, specific and sensitive detec-tion of human seasonal influenza virus ( H1, H3 and B) by using molecular biological and immunological methods in combination.Methods The primers were designed according to the genes encoding the matrix protein ( M) , the H1 and H3 hemagglutinin ( HA) of influenza A virus and the nonstructural proteins ( B-NS) of influenza B virus and then were labeled with biotin.The PCR products were detected by ELISA by use of an internal catching probe labeled with DIG.Results The minimum copy numbers of genes encoding the M, H1, H3 and B-NS proteins detected by the established assay were 1.43?103 , 8.67?102 , 3.86?103 and 5.45?103 copies/μl, respectively, which indicated that the PCR-ELISA assay was about 10 times more sensitive than agarose gel electrophoresis in the detection of PCR products.No cross-reactions between the different subtypes of influenza virus or different species of virus were observed.Moreover, a total of 104 clin-ical specimens of influenza virus were examined by the PCR-ELISA assay, the results of which were consist-ent with those of the virus isolation method.Conclusion The newly developed PCR-ELISA assay was a highly sensitive and specific method for the rapid detection and subtyping of influenza virus, suggesting the possibility of using it in laboratory for the surveillance and detection of influenza virus.

Objective To study the characteristics of MRI Lesions in posterior circulation cerebral infarction and the correlation of the syndrome types of traditional Chinese medicine.Methods 82 patients with posterior circulation cerebral infarction from May 2012 to May 2015 were recruited, two observers estimated cranial MRI infarct size, location, and TCM syndrome type in the hospital medical records of the patients separately.Results Of all patients with posterior circulation cerebral infarction, patients with meridians involved infarction in the distant part of posterior circulation is far more than other parts of the posterior circulation(21 patient,P=0.006), nevertheless,patients with apoplexy involving Zang-fu organs,cerebral infarction volume is larger (14.78 ± 5.68 mlvs. 9.12 ± 6.67 ml,P=0.001).Conclusion Of all patients with posterior cerebral infarction, patients with meridians be involved, infarction in the distant part of posterior circulation is far more than other parts of the posterior circulation, nevertheless, in patients with apoplexy involving Zang-fu Organs, cerebral infarction volume is larger.

A chemiluminescence enzyme immunoassay based on magnetic microparicles (MmPsCLEIA) was developed to evaluate serum α-fetoprotein (AFP) in parallel with tramional colorimetric enzyme-linked immunsorbrnt assay (ELISA).A sestematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer of immunoreagents,less total assay time,and better linearity,recovery,precision,senitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results werecompared with commercial electrochemilunminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R2=0.6703; however,the correlation between MPs-CLEIA and ECLIA (R2=0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R2=0.6866).

Glypican-3 (GPC3) is reported as a great promising tumor marker for hepatocellular carcinoma (HCC) diagnosis.Highly sensitive and accurate analysis of serum GPC3 (sGPC3),in combination with or instead of traditional HCC marker alpha-fetoprotein (AFP),is essential for early diagnosis of HCC.Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay.Here,the magnetic nanoparticles (MnPs) and magnetic microparticles (MmPs) with carboxyl groups were further modified with streptavidin,and applied for the development of chemiluminescence enzyme immunoassay (CLEIA).After comparing between MnPs- and MmPs-based CLEIA,MnPs-based CLEIA was proved to be a better method with less assay time,greater sensitivity,better linearity and longer chemiluminescence platform.MnPs-based CLEIA was applied for detection of sGPC3 in normal liver,hepatoeirrhosis,secondary liver cancer and HCC serum samples.The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

A simple assay for detection of phospholipase C (PLC) activity was developed based on a fluorescence liposome probe using the Liss Rhod PE-loaded phospholipid liposomes.The liposome probe was prepared by the coassembly of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and fluorescent lipid (Liss Rhod PE).The probe showed very low background fluorescence due to fluorescence self-quenching effect of Liss Rhod PE.As the PLC enzyme selectively digested lipid, the Rhod fluorescence was recovered from its quenched state, leading to the sensitive detection of PLC.This assay provided a limit of detection (at a signal-to-noise ratio of 3) of 2 U/L for PLC.In the presence of PLC inhibitor, the fluorescent response of the sensor for PLC decreased, indicating that the assay could also be used for screening PLC inhibitors.

Glypican-3 （GPC3） is reported as a great promising tumor marker for hepatocellular carcinoma （HCC） diagnosis. Highly sensitive and accurate analysis of serum GPC3 （sGPC3）, in combination with or instead of traditional HCC marker alpha-fetoprotein （AFP）, is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles （MnPs） and magnetic microparticles （MmPs） with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay （CLEIA）. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

Objective: To investigate the characteristics of 25(OH)D level in children with ASD and its correlation with clinical features. Methods: A total of 196 children with ASD who received outpatient and inpatient rehabilitation training from January 2017 to January 2019 were included in ASD group, and 178 healthy children who visited the hospital during the same period were included in healthy control group. Differences in 25(OH)D levels and general data between study group and healthy control group were compared. In addition, ASD group was divided into 25(OH)D normal group and abnormal group in accordance with 25(OH)D level (≥30 ng/mL). Differences in general data, total score of CARS scale and factor scores were compared between two groups. Finally, the correlation between serum 25(OH)D level and CARS total score and factor scores of children with ASD was evaluated. Results: 25(OH)D level in ASD group was significantly lower than that in healthy control group (P<0.01). The incidence of sleep disorder, dietary bias, vomiting, constipation and diarrhea in children with ASD was statistically significant compared with that of healthy children (P<0.01); there were statistically significant differences in breastfeeding, sleep disorder, dietary bias and diarrhea between 25(OH)D normal group and abnormal group (χ2=4.97，8.69,6.67,3.98,P<0.05); there were statistically significant differences in 10 aspects including CARS total score, interpersonal relationship, imitation, emotional response, physical use ability, relationship with inanimate objects, adaptation to environmental changes, visual response, auditory response and general impression (P<0.05); there was a significant negative correlation between serum 25(OH)D level and CARS total score in children with ASD (r=-0.32, P<0.01). Conclusion Breastfeeding could reduce the risk of 25(OH)D abnormalities in children with ASD. 25(OH)D reduction would cause sleep disorder, dietary bias and gastrointestinal problems, while dietary bias and gastrointestinal problems would affect 25(OH)D uptake and absorption. 25(OH)D might be related to the occurrence of ASD in children. Serum 25(OH)D level could be used as a reference index for the severity of ASD in children.

Objective To investigate the clinical efficacy of neoadjuvant chemotherapy combined with radical gastrectomy for advanced gastric cancer.Methods The retrospective cross-sectional study was conducted.The clinicopathological data of 73 patients who underwent neoadjuvant chemotherapy combined with radical gastrectomy for advanced gastric cancer at the First Affiliated Hospital of Zhejiang University between June 2004 and December 2009 were collected.Neoadjuvant chemotherapy regimens included XELOX and FOLFOX.Patients received radical gastrectomy within 2 weeks after the completion of the last cycle of neoadjuvant chemotherapy and then continued to undergo postoperative neoadjuvant chemotherapy.Observation indicators:(1) adverse event of neoadjuvant chemotherapy;(2) surgical and postoperative situations;(3) follow-up situations.Follow-up using outpatient examination and telephone interview was performed to detect survival of patients up to December 2014.Measurement data with skewed distribution were described as M (range).Overall survival time was from the beginning of treatment to death or end of follow-up (patients with loss to follow-up).Progression-free survival time was from the beginning of treatment to tumor progression,recurrence and metastasis or death.The survival curve was drawn by the Kaplan-Meier method.Results (1) Adverse event of neoadjuvant chemotherapy:of 73 patients,38 received XELOX regimens and 35 received FOLFOX regimens,with a median cycle of 3 (range,1-7 cycles).There were 55 adverse events during neoadjuvant chemotherapy,including 47 with grade 1-2 and 8 with grade 3-4.(2) Surgical and postoperative situations:all the 73 patients underwent successful D2 radical gastrectomy for gastric cancer,including 40 receiving total gastrectomy,31 receiving distal gastrectomy,1 receiving total gastrectomy with transverse colon resection and 1 receiving distal gastrectomy with cholecystectomy.Of 73 patients,10 with postoperative complications were improved by conservative treatment,including 3 with pleural effusion,2 with peritoneal effusion,2 with anastomotic bleeding,2 with cholecystitis and 1 with lympha fistula.No patient received reoperations or died within 30 days postoperatively.Pathological TNM staging:22 patients were detected in stage Ⅰ-Ⅱ,45 in stage Ⅲ,4 in stage Ⅳ and 2 in stage T0N1M0.Three patients (in stage T0N0M0) had complete remission.Forty-three patients underwent postoperative chemotherapy.(3) Followup:all the 73 patients were followed up for 8-125 months,with a median time of 51 months.The median survival time,5-year overall survival rate and 5-year disease-free survival rate of 73 patients were 52 months,41.1％ and 34.2％,respectively.Conclusion XELOX and FOLFOX regimens of neoadjuvant chemotherapy combined with radical gastrectomy for advanced gastric cancer are safe and effective.

Objective This article introduced the concrete methods and benefits of promoting and holding competition of acupuncture and moxibustion in TCM universities or colleges. A regular skill competition will not only stimulate students' passion for TCM study and expand teaching methods, but also serve as a effective way to enhance practical skills of students.

OBJECTIVETo study the alkaloids in the stems and leaves of Stephania cepharantha.

METHODThe dried stems and leaves of S. cepharantha were percolated with 95% ethanol and the solvent was removed by rotary evaporation to give a concentrate, and the concentrate was extracted by petroleum ether and chloroform. Column chromatograghy on MCI CHP 20P, silica gel, Rp-18, Sephadex LH-20 and polyamide were applied for the isolation and purification of the chloroform fraction. The structures were elucidated by their physicochemical properties and spectral data.

RESULTEleven alkaloids were obtained and identified as lysicamine (1), tetrahadropalmatine (2), palmatine (3), isocorydione (4), corydalmine (5), corypalmine (6), sinoracutine (7), sinoacutine (8), cepharamine (9), isocorydine (10) and corydine (11).

CONCLUSIONCompounds 2-7 were isolated from S. cepharantha for the first time, and compound 7 was isolated from the genus Stephania for the first time, compound 4 was isolated from the Menispermaceae family for the first time.