A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.
Colorimetry ; methods ; Coronavirus Infections ; diagnosis ; virology ; Coronavirus NL63, Human ; genetics ; isolation & purification ; DNA Primers ; genetics ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Reverse Transcription ; Sensitivity and Specificity

Objective To study the influence of postnatal high protein feeding on learning and memory abilities of preterm rats.Methods After weaning,the SD offspring were divided into 6 groups randomly according random digits table:preterm standard protein group (group A),term standard protein group (group B),preterm low protein group (group C),term low protein group (group D),preterm high protein group (group E),and term high protein group (group F),with 34 rats in each group.Starting from 22 days of age,the standard protein group,low-protein group and the high-protein group were respectively fed with a standard protein diet (containing 18％ protein),low-protein diet (containing 8％ protein) and a high-protein diet (containing 30％ protein) until the end of experiment.Within each group,the rats were respectively tested in 6 weeks and 8 weeks.Morris water maze task was performed to assess the learning and memory abilities of the premature rats.Results 1.Directional navigation experiments:(1) At the age of 6 weeks,the escape latencies to find the platform were shortened with increased training times for full-term rats.The escape latencies of the premature rats did not change significantly on the third and fourth day of the experiment.(2) At the age of 8 weeks,the escape latencies to find the platform were shortened with increased training time for all of the rats.2.Probe trial test:(1)At the age of 6 weeks,premature rats spent significantly less time in target quadrant than full-time rats.And the percent age of traveled distance in target quadrant of premature rats was significantly less than full term rats (P ＜ 0.05).(2) At the age of 8 weeks,group E [(68.48 ± 20.65) s] spent significantly greater time in target quadrant than group A and group C [group A(52.41 ± 11.24) s,group C (49.54 ± 11.04) s].And the percent age of traveled distance in target quadrant of group E [(36.56 ± 10.44) ％] were significantly higher than group A and group C [group A (29.83 ± 8.33) ％,group C (26.97 ± 5.41) ％] (P ＜ 0.05).(3) At the age of 8 weeks,group E spent time in target quadrant and the percent of traveled distance in target quadrant [(68.48 ± 20.65) s,(36.56 ±10.44)％] were higher than group F [(61.29 ± 16.27) s,(35.71 ±6.61)％],but there was no significant difference (P ＞ 0.05).Conclusions In the early growth and development process of the brain,premature birth may seriously affect early brain functions in rats,and the learning and cognitive abilities of the premature rats are worse than full-term rats.In the subsequent process of growth and development,after a long time of high protein nutritional intake,the learning and cognitive abilities of the premature rats can be restored and they can catch up with full-term rats.

OBJECTIVETo detect the heterogeneity of mitochondrial DNA (mtDNA) in black and white hair of patients with type 2 diabetes.

METHODSMtDNA was extracted from the hair shaft of the patients to amplify two target DNA fragment from mtDNA coding region and control region using PCR. The differences in the heterogeneity in the target DNA fragment was analyzed between diabetic patients and the control group with denaturing high-performance liquid chromatography (DHPLC).

RESULTSIn the control subjects and diabetic patients, the mtDNA heterogeneity in the black hair was 3% and 10% in 20-45 year-old groups and 9% and 17% in 45-70 year-old groups, as compared to 9%, 20%, 21%, and 40% in the white hair, respectively. The mtDNA heterogeneity in the black and white hair was both higher in the diabetic patients than in the control subjects of the same age group, and was also higher in older age subgroups in both control and diabetic groups (P<0.05). The white hair mtDNA showed a significantly higher heterogeneity than the black hair mtDNA in the two age groups of diabetic patients and in 45-70 year-old control group (P<0.05).

CONCLUSIONThe mtDNA heterogeneity in the hair increases in type 2 diabetic patients and show an association with aging.

Adult ; Age Factors ; Aging ; genetics ; Chromatography, High Pressure Liquid ; methods ; DNA, Mitochondrial ; genetics ; Diabetes Mellitus, Type 2 ; genetics ; metabolism ; Female ; Genetic Heterogeneity ; Hair ; metabolism ; Humans ; Male ; Middle Aged ; Young Adult

Objective To study the influence of protein intake in learning and memory capabilities and mammalian target of rapamycin (mTOR) expression in premature rats.Methods Premature rars were randomly divided into three groups after weaning:preterm standard protein group,preterm low protein group and preterm high protein group.After weaning,three groups were respectively fed with standard protein diets (18％ protein),low protein diets (8％ protein) and high protein diets (30％ protein) until the end of experiment.When rats were at the 6th and 8th weeks,they were performed Morris water maze test to assess the learning and memory abilities,and irnmunohistochemistry and Westem blotting were used to observe the mTOR distribution and mTOR protein expression in the hippocampus.Results At the age of 6 weeks,the escape latencies of the premature rats did not change significantly on the 3rd and 4th day of the experiment; the frequency of crossing the platform and the mTOR-positive cells in hippocampal CA1 and CA3 regions of each group showed no significant differences (P＞0.05).At the age of 8 weeks,the escape latencies in finding the platform were shortened with increased training times for premature rats; the frequency of crossing the platform of high-protein premature rats was significantly higher than that in the other two groups (P＜0.05); and the mTOR expression in the hippocampal CA1 and CA3 regions of high-protein premature rats was also significantly increased as compared with the other two groups (P＜0.05).High-protein premature rats at the age of 8 weeks had significantly higher frequency of crossing the platform and increased mTOR-positive cells in the hippocampal CA1 and CA3 regions than those at the age of 6 weeks (P＜0.05).Western blotting also indicated that the hippocampal mTOR protein expression was not significantly different in each group at 6 week-old; the mTOR expression of high-protein premature rats was significantly higher than that in the other two groups at the age of 8 weeks.Conclusions In the early growth and development process of the brain,mTOR activity might be inhibited in the premature birth rats,which can lead to impaired learning and memory abilities.After a long time of high protein nutritional intake,impaired learning and cognitive abilities of premature rats can get recovery,which may be associated with up-regulation ofmTOR expression.

Objective:To investigate the application value of P-loop digestive tract recons-truction in pancreaticoduodenectomy (PD).Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 21 ampullary disease patients undergoing PD in the Liuzhou People′s Hospital Affiliated to Guangxi Medical University from April to December 2020 were collected. There were 13 males and 8 females, aged from 35 to 76 years, with a median age of 60 years. All the 21 patients underwent PD and digestive tract reconstruction using P-loop method based on the Child reconstruction. Observation indicators: (1) surgical situations; (2) postoperative situations; (3) follow-up. Follow-up was conducted using outpatient examination or telephone interview to detect survival and discomfort symptoms of patients up to December 2020. Measurement data with normal distribution were represented as Mean± SD, and measurement data with skewed distribution were represented as M(range). Count data were described as absolute numbers or persentages. Results:(1) Surgical situations: all the 21 patients underwent PD successfully. The operation time, time of P-loop anastomosis and volume of intraoperative blood loss of 21 patients were (317±74)minutes, (14±3)minutes and 375 mL(range, 100-800 mL), respectively. Of the 21 patients, 17 cases had pancreatic texture as soft, 4 cases had pancreatic texture as hard, 3 cases had diameter of pancreas ≤3 mm, 18 cases had diameter of pancreas >3 mm, 14 cases were placed pancreatic duct stent, 7 cases were not placed pancreatic duct stent. (2) Postoperative situations: 2 of the 21 patients had grade A pancreatic fistula, and none of patient had grade B or grade C pancreatic fistula. One case had hepaticojejunal anastomotic fistula, 2 cases without pancreatic fistula had delayed gastric emptying and none of patient had abdominal infection or bleeding. The duration of postoperative hospital stay of 21 patients was (16±5)days, and none of patient died during postoperative 30 days. Results of postoperative histopathological examination showed there were 10 cases with duodenal papillary carcinoma, 4 cases with lower bile duct carcinoma, 3 cases with pancreatic head ductal adenocarcinoma, 1 case with duodenum stromal tumors, 1 case with gastric antrum carcinoma, 1 case with mass in the head of the pancreas of IgG4 and 1 case with choledochal cyst of type 3. (3) Follow-up: all 21 patients were followed up for 1.0 to 7.0 months, with a median follow-up time of 4.3 months. None of patient died. There was no abdominal pain, distension or dyspepsia during follow-up. One case was diagnosed as tumor liver metastasis at postoperative 5 months.Conclusion:P-loop digestive tract reconstruction in PD is safe and effective, with good short-term effect.