Objective To establish a rapid detection method for zika virus based on direct amplification re-al-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)technique.Methods A direct amplification RT-PCR technique for the rapid detection of zika virus in 5 samples(whole blood,serum,saliva,throat swab and urine)was established by using a special function DNA polymerase and a preferred PCR enhancer.Results The detection limits of the 5 samples were 103 PFU/mL in serum,102 PFU/mL in urine,throat swab,and saliva,and 104 PFU/mL in whole blood.The coefficient of goodness-fit of stand-ard curves was above 0.98,and the amplification efficiency was 90％-110％.Zika virus nucleic acid was suc-cessfully amplified,but non-zika virus nucleic acid was not amplified.Based on the repeatable detection of sam-ples from urine,whole blood,and saliva,the variation coefficient of 6 repeated Ct values at 106 PFU/mL and 102 PFU/mL concentrations were all＜5％.The zika virus detection method established by the direct amplifi-cation RT-PCR technique was consistent with the detection results of conventional RT-PCR technique.Only two serum samples were detected in eight zika virus samples,and the remaining 62 non-zika virus samples and 12 negative samples were not amplified.Conclusion A rapid detection method for zika virus based on direct ampli-fication RT-PCR technique is successfully established.The method is simple,rapid,sensitive and specific.

To analyze the correlation of ADH4 exon rs1126671 and ADH7 exon rs971074 polymorphisms with risky drinking behaviors and alcoholic liver disease. The patients with alcoholic liver disease diagnosed in the Gastroenterology Department of the People′s Hospital of Hechi from November 2021 to June 2022, including 52 cases of alcoholic liver disease with positive risky drinking behaviors, 103 cases of non-alcoholic liver disease with positive risky drinking behaviors of the same gender and age, and 105 healthy subjects with no risky drinking behaviors as control groups were retrospectively analyzed. The serum total protein and albumin are detected by immunoturbidimetry and globulin is calculated by the difference method; the serum total bilirubin and direct bilirubin are detected by the nitrite oxidation method and indirect bilirubin is calculated by the difference method; alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and γ-glutamyl transferase are detected by the substrate method. The results revealed that all 52 patients with alcoholic liver disease were male. The non-parametric independent sample Kruskal-Wallis test was adopted to analyze the baseline of twelve liver functions among the alcoholic liver disease group, the risky drinking behavior group and the healthy control group, and it was found there was statistical significance in ten major liver function indicators in the difference comparison among the three groups like serum total protein (g/L) 65.0 (60.1, 71.4), 73.4 (70.3, 76.3), 72.4 (69.2, 76.2) ( H=37.130, P<0.001); albumin (g/L) 36.1 (28.6, 42.9), 47.2 (45.0, 49.2), 47.5 (45.9, 49.5) ( H=14.503, P=0.001); direct bilirubin (μmol/L) 10.1 (35.6, 34.0.1), 3.8 (3.1, 5.45), 4.2 (2.9, 6.0) ( H=26.608, P<0.001); alkaline phosphatase (U/L) 106.0 (71.0, 164.0), 68.0 (57.5, 82.0), 70.0 (59.0, 87.0) ( H=27.904, P<0.001); albumin to globulin 1.34 (0.91, 1.88), 1.82 (1.65, 2.00), 1.89 (1.68, 2.07) ( H=11.047, P=0.004); direct bilirubin to indirect bilirubin 0.91 (0.69, 1.91), 0.41 (0.35, 0.54), 0.42 (0.34, 0.54) ( H=19.478, P<0.001); serum total bilirubin (μmol/L) 23.9 (13.7, 51.0), 13.8 (10.2, 17.9), 13.0 (10.1, 17.4) ( H=18.375, P<0.001); aspartate aminotransferase (U/L) 74.0 (39.0, 122.0), 22.0 (19.0, 28.0), 23.0 (19.0, 30.0) ( H=76.365, P<0.001); alanine aminotransferase (U/L) 37.0 (25.0, 55.0), 23.0 (17.0, 30.0), 24.0 (17.0, 33.8) ( H=57.041, P<0.001); γ-glutamyl transferase (U/L) 135.0 (45.0, 364.0), 33.0 (23.5, 49.5), 32.0 (19.0, 49.0) ( H=82.558, P<0.001); however, there were no statistical significance in the pairwise comparisons between risky drinking and healthy groups. The two loci of ADH4 and ADH7 were in genetic linkage equilibrium. In the three groups of samples, the ADH4 gene rs1126671 locus was comprised primarily of the CC homozygous genotype, and there was no TT genotype. The ADH7 gene rs971074 genotype had statistical difference in the comparison of the three groups ( χ2=9.370, P<0.05). Compared with the CC genotype, the CT genotype had no statistical difference in the pairwise comparison between the risky drinking behavior group and alcoholic liver disease group, and the healthy group and alcoholic liver disease group. There was a statistical difference in that between the healthy group and the risky drinking behavior group ( χ2=6.372, P=0.012). The analysis display of mode of inheritance between RD group and HA group was statistically significant in the difference of the superdominance inheritance mode ( OR=2.92, 95% CI:1.22-6.98; P=0.012), the dominant inheritance mode ( OR=2.90, 95% CI:1.26-6.64; P=0.008), the co-dominant inheritance mode ( OR=2.96, 95% CI:1.24-7.08; P=0.032) and the additive mode ( OR=2.46, 95% CI:1.16-5.22; P=0.013). In general, the CT genotype of ADH7 gene rs971074 is a risk factor for positive risky drinking behavior, and the ADH family may still increase the susceptibility of people with a potential alcoholic liver disease protection background through the correlation between ADH7 and risky drinking behavior.

To analyze the correlation of ADH4 exon rs1126671 and ADH7 exon rs971074 polymorphisms with risky drinking behaviors and alcoholic liver disease. The patients with alcoholic liver disease diagnosed in the Gastroenterology Department of the People′s Hospital of Hechi from November 2021 to June 2022, including 52 cases of alcoholic liver disease with positive risky drinking behaviors, 103 cases of non-alcoholic liver disease with positive risky drinking behaviors of the same gender and age, and 105 healthy subjects with no risky drinking behaviors as control groups were retrospectively analyzed. The serum total protein and albumin are detected by immunoturbidimetry and globulin is calculated by the difference method; the serum total bilirubin and direct bilirubin are detected by the nitrite oxidation method and indirect bilirubin is calculated by the difference method; alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and γ-glutamyl transferase are detected by the substrate method. The results revealed that all 52 patients with alcoholic liver disease were male. The non-parametric independent sample Kruskal-Wallis test was adopted to analyze the baseline of twelve liver functions among the alcoholic liver disease group, the risky drinking behavior group and the healthy control group, and it was found there was statistical significance in ten major liver function indicators in the difference comparison among the three groups like serum total protein (g/L) 65.0 (60.1, 71.4), 73.4 (70.3, 76.3), 72.4 (69.2, 76.2) ( H=37.130, P<0.001); albumin (g/L) 36.1 (28.6, 42.9), 47.2 (45.0, 49.2), 47.5 (45.9, 49.5) ( H=14.503, P=0.001); direct bilirubin (μmol/L) 10.1 (35.6, 34.0.1), 3.8 (3.1, 5.45), 4.2 (2.9, 6.0) ( H=26.608, P<0.001); alkaline phosphatase (U/L) 106.0 (71.0, 164.0), 68.0 (57.5, 82.0), 70.0 (59.0, 87.0) ( H=27.904, P<0.001); albumin to globulin 1.34 (0.91, 1.88), 1.82 (1.65, 2.00), 1.89 (1.68, 2.07) ( H=11.047, P=0.004); direct bilirubin to indirect bilirubin 0.91 (0.69, 1.91), 0.41 (0.35, 0.54), 0.42 (0.34, 0.54) ( H=19.478, P<0.001); serum total bilirubin (μmol/L) 23.9 (13.7, 51.0), 13.8 (10.2, 17.9), 13.0 (10.1, 17.4) ( H=18.375, P<0.001); aspartate aminotransferase (U/L) 74.0 (39.0, 122.0), 22.0 (19.0, 28.0), 23.0 (19.0, 30.0) ( H=76.365, P<0.001); alanine aminotransferase (U/L) 37.0 (25.0, 55.0), 23.0 (17.0, 30.0), 24.0 (17.0, 33.8) ( H=57.041, P<0.001); γ-glutamyl transferase (U/L) 135.0 (45.0, 364.0), 33.0 (23.5, 49.5), 32.0 (19.0, 49.0) ( H=82.558, P<0.001); however, there were no statistical significance in the pairwise comparisons between risky drinking and healthy groups. The two loci of ADH4 and ADH7 were in genetic linkage equilibrium. In the three groups of samples, the ADH4 gene rs1126671 locus was comprised primarily of the CC homozygous genotype, and there was no TT genotype. The ADH7 gene rs971074 genotype had statistical difference in the comparison of the three groups ( χ2=9.370, P<0.05). Compared with the CC genotype, the CT genotype had no statistical difference in the pairwise comparison between the risky drinking behavior group and alcoholic liver disease group, and the healthy group and alcoholic liver disease group. There was a statistical difference in that between the healthy group and the risky drinking behavior group ( χ2=6.372, P=0.012). The analysis display of mode of inheritance between RD group and HA group was statistically significant in the difference of the superdominance inheritance mode ( OR=2.92, 95% CI:1.22-6.98; P=0.012), the dominant inheritance mode ( OR=2.90, 95% CI:1.26-6.64; P=0.008), the co-dominant inheritance mode ( OR=2.96, 95% CI:1.24-7.08; P=0.032) and the additive mode ( OR=2.46, 95% CI:1.16-5.22; P=0.013). In general, the CT genotype of ADH7 gene rs971074 is a risk factor for positive risky drinking behavior, and the ADH family may still increase the susceptibility of people with a potential alcoholic liver disease protection background through the correlation between ADH7 and risky drinking behavior.

Objective:To investigate the effect of Asarinin on the survival time of transplanted heart after allogeneic heterotopic heart transplantation and to further verify the anti-immune rejection effect of Asarinin in spleen and peripheral blood.Methods:Using 64 Wistar rats as donors, 64 SD rats as recipients to establish the allogeneic heterotopic heart transplantation model in rats.After successful transplantation, 64 rats were use simple randomization divided into control group, cyclosporine A(CsA) group, Asarinin group and half CsA + half Asarinin group with 16 rats in each group.CsA group was given 5 mg/kg by gavage; Asarinin group was given 25 mg/kg; half dose group was given CsA 2.5 mg/kg+ Asarinin 12.5 mg/kg and the control group was given the same volume of normal saline by gavage.After administration for 1 week, half of them were used to observe the survival time.The other half of the rats were fully anesthetized with chloral hydrate, spleen and peripheral blood were taken.Half of the spleen was taken to observe the slices under the microscope.The other half of spleen was used RT-PCR to detect the relative expression of IFN-γ and IL-4.The expression of co-stimulatory molecules CD80, CD86 and CD40 in peripheral blood were detected by flow cytometry.Results:Survival time of transplanted heart was control group (8.4±0.9), CsA group (30.5±8.3), Asarinin group (16.5±4.3) and half-dose group (26.1±5.2) days.Compared with control group, survival time of heart transplantation became prolonged in all groups and the difference was statistically significant ( P<0.05). HE staining of splenic tissue showed that, as compared with control group, the injury of each group was alleviated.The relative expression of IFN-γ in spleen was control group (1.055±0.083), CsA group (0.396±0.038), Asarinin group (0.833±0.094) and half-dose group (0.862±0.104). The last three groups were lower than control group and the difference was statistically significant ( P<0.05). The relative expression of IL-4 in spleen was control group (1.429±0.234), CsA group (3.808±0.729), Asarinin group (2.209±0.306) and half-dose group (2.323±0.321). The last three groups all spiked as compared with control group and the difference was statistically significant ( P<0.05). The expressions of CD80, CD86 and CD40 in peripheral blood were control group (98.21±0.54), (85.78±0.89) and (96.36±0.66), CsA group (89.26±0.36), (56.86±2.32) and (88.11±1.61), Asarinin group (94.19±0.47), (79.01±1.12) and (87.86±1.67) and half-dose group (94.87±0.74), (80.81±0.98) and (89.71±0.97) respectively.The last three groups were lower than control group and the difference was statistically significant ( P<0.05). Conclusions:Asarinin can prolong the survival time of transplanted heart after allogeneic heterotopic heart transplantation in rats, inhibit the immune injury of spleen after allogeneic heterotopic heart transplantation in rats, decrease IFN-γ in spleen, increase IL-4 in spleen and inhibit the expression of peripheral blood costimulatory molecules CD80, CD86 and CD40.

Imported malaria has become a major risk factor for malaria prevention and control in China. How to screen malaria quickly for people entering China is an urgent problem to be solved. Protein microarrays are widely used in high-throughput screening and diagnosis. In this study, surface plasmon resonance (SPR) technique for malaria detection was established by using the specific adsorption surface treated by polyethylene glycol polymer, and the malaria specific antigen HRP2 was used as capture probe. The optimal concentration of antigen, sensitivity and specificity of detection, as well as anti-interference ability of the chip were analyzed. The SPR protein chip was applied to detect specific antibodies of malignant malaria in serum with the advantage of label-free, instant and fast. Compared with fluorescence quantitative PCR, there were no significant difference in sensitivity and specificity between the two methods. This study lays a foundation for further development of protein microarray for malaria typing identification, and it is conducive to the rapid screening of malaria for people entering.
Antibodies ; China ; Humans ; Malaria/diagnosis* ; Protein Array Analysis ; Surface Plasmon Resonance

The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.
Adsorption ; Autoantibodies ; Dimethylpolysiloxanes ; Protein Array Analysis ; Silica Gel ; Surface Properties

Objective To explore the clinical value of magnetic resonance diffusion-weighted imaging ( MR-DWI ) in the early diagnosis of cervical lymph node recurrence after radiotherapy of nasopharyngeal carcinoma, aiming to provide reference for targeted diagnosis and treatment of these patients. Methods The MR-DWI features of 17 patients with recurrent cervical lymph nodes after radiotherapy from 2005 to 2016 were retrospectively analyzed. The results of diagnosis and treatment after lymph node recurrence were summarized. Results The recurrent lymph nodes of 17 patients showed a high signal or mixed signal on MR-DWI images. The sensitivity of MR-DWI and T2WI fat suppression sequence was 100% and 60%. Positron emission tomography-computed tomography ( PET-CT) or biopsy was performed to validate the diagnosis in patients with highly suspected single cervical recurrence. Besides, surgical treatment yielded better clinical prognosis. Conclusions MR-DWI is highly sensitive to recurrent cervical lymph nodes of nasopharyngeal carcinoma after radiotherapy, especially for the small lymph nodes of 5-10 mm in diameter, which are easily ignored. PET-CT examination should be performed, the nature of the lymph nodes should be confirmed by multi-modality imaging diagnosis, and timely operation has important clinical significance in improving the therapeutic effect and quality of life for patients with cervical lymphnode recurrence.

Objective To study the feasibility of detecting fetus RhD type gene by Surface Plasmon Reso-nance(SPR)technology,and to establish a new rapid diagnosis method for fetus RhD type gene.Methods The different types of DNA corresponding RNA probes were fixed on the surface of SPR chip by using amino cou-pling methods,and optimize the chip analysis condition,and then using the RNase H enzyme hydrolysis,signal amplification detection,at last the detection conditions were determined.We use the RhD type gene exon 5,7 of RNA probe to test its corresponding DNA molecules,and analyse the specificity and sensitivity of SPR chip detection signal.Results The SPR technique for detecting the exon 5,7 of RhD blood type gene shows good sensitivity and specificity in all,SPR technology can specifically detect the Exon 5,7 of RhD blood type gene, and the sensitivity of for detecting RhD gene exon 5,7 is 100 pmol/L by SPR.Conclusion The SPR technolo-gy can quickly detect RhD gene accordingly,SPR technology is simple,rapid,reliable and label-free,w hich can provide a new way predicting fetal RhD type for RhD negative prenatal.

Objective To clarify the diagnostic value of diffusion-weighed imaging ( DWI) in the medial group of retropharyngeal lymph nodes in nasopharyngeal carcinoma, understand the clinical characteristics of retropharyngeal lymph nodes and explore the feasibility of optimizing the target volume of CT V60. Methods Clinical data of 437 patients with clinical stage Ⅰ-IVa nasopharyngeal carcinoma admitted to Jiangsu Cancer Hospital from 2011 to 2017 were retrospectively analyzed. All patients underwent magnetic resonance imaging (MRI),DWI (1 000 s/mm2) and enhanced CT scans to analyze the clinical characteristics of retropharyngeal lymph nodes and investigate the dosimetric advantage and safety of CT V60 lower margin on the upper margin of C2. Results The medial lymph nodes with a transverse diameter of 2. 0-19. 0 mm were detected 13 of 437 patients,and 53. 8% of the lymph nodes were measured 2-5 mm in transverse diameter. The medial lymph nodes were distributed between the superior margin of C1and 1/3 of C3.Its occurrence was related to N stage,double cervical lymph node metastases,especially the transverse diameter of cervical lymph node＞ 3 cm.The sensitivity of DWI,T2and enhanced CT were 100%,61. 5% and 23. 1%.After the special cases were excluded,the lower margin of CT V60on the superior margin of C2was separated. The radiation dose and volume of the swallowing structures were significantly decreased. The 5-year survival rate was 80% without recurrence in the optimized region. Conclusions The incidence of the medial group of retropharyngeal lymph nodes is low with a diameter of less than 5 mm. DWI possesses advantages in displaying the medial group of retropharyngeal lymph nodes. Isolating the lower margin of CT V60from the superior margin of C2is safe and feasible and has dosimetric advantages for protecting swallowing structure.

Objective To develop a biosensor method with strong specificity and high‐throughput by combining with the surface plasmon resonance (SPR) and gene chip technique and aiming at 9 kinds of common respiratory tract viruses including influenza A and influenza B ,(Influ A ,B) ,H1N1 ,respiratory syncytial virus (RSV) ,parainfluenza virus 1 -3 (PIV1 -3) ,adenovirus (ADV) and coronavirus (SARS) leading to severe acute respiratory syndrome .Methods Firstly the software primer 5 was used to design the specific primer and probe of related viruses in the conserved sequence ;the designed nine kinds of corresponding respiratory virus probes were immobilized in the specific region of SPR chip after chemical modification .The SPR technique was applied to conduct the real time monitoring the hybridization process of the probe with the PCR products .Finally the signal amplification was realized by the biotin and streptavidin system .Results The designed gene chip could detect 9 kinds of respiratory tract viruses by high‐throughput with better detection specificity ;the chip surface could be reutilized after certain regeneration condition ,which avoided the influence of intra‐batch difference on the results ;the detection sensitivity reached the nanomole level .Conclusion The prelimi‐nary study results demonstrate that using the SPR biosensor technique to establish a high‐‐throughput detection of respiratory tract viruses has some practicability and feasibility ,and is expected to become a rapid ,large scale and high‐ throughput measure for screening respiratory tract viruses with good application prospect .