Establishment and preliminary application of dual fluorescent quantitative PCR for detection of RV and CAV2 / 公共卫生与预防医学

Objective To investigate the shedding of CAV2-ΔE3-CGS after immunization and the background of canine adenovirus (CAV) infection, and to establish a dual fluorescent quantitative PCR detection method for rabies virus (RV) and canine adenovirus type 2 (CAV2). Methods A dual fluorescent quantitative PCR detection method was established by designing specific primers and probes for E1 gene of CAV and G gene of RV for the detection of CAV2-ΔE3-CGS. Oral swabs, anal swabs and environmental samples of stray dogs from experimental animal farm and dog detention center were tested. Results The standard curves generated by this method were Y=-3.351 × logX + 44.895, R2 = .999 and Y=-3.413 × logX + 45.192, R2=0.996, respectively. The linear relationships were good, and the minimum detection limits were both 102 copies/μL. CAV2-ΔE3-CGS was not detected in experimental animal farm. CAV was detected in dog detention center, and the positive rates were 5.88% (5/85) in oral swabs, 8.24% (7/85) in anal swabs, and 4% (1/25) in environmental samples. Conclusion The dual fluorescent quantitative PCR method can be used for the detection of CAV2-ΔE3-CGS after immunization and the investigation of CAV infection. The present study has shown that no CAV2-ΔE3-CGS has been detected after immunization and CAV infection rate of stay dogs is low in Shanghai. CAV2-ΔE3-CGS oral immunization meets requirement and is applicable.

Establishment and evaluation of a method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples / 中国血吸虫病防治杂志

Objective To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. Methods The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers’ urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). Results An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers’ urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). Conclusions A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.

Molecular quantification for quality control of Jinqian Baihuashe in Jinlong Capsule / 药学学报

The quality control of Chinese patent medicines containing animal-derived crude drugs is relatively difficult, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are relevant methods, they are usually qualitative, and quantitative indicators are either lacking or have poor specificity. This paper has proposed to use molecular quantitative technology to control the quality of Chinese patent medicines containing animal-derived crude drugs. In this study, a molecular quantitative method based on fluorescence quantitative PCR was established for the determination of Jinqian Baihua She in Jinlong Capsule. The method has good specificity, sensitivity, and repeatability. There is a good linear relationship between the content of DNA fragments and the CT (cycle threshold) value. The content of the Bungarus multicinctus-specific fragment in Jinlong Capsule is 24.1-46.6 IU·mg-1. It is suggested that the content of the specific fragment of Jinqian Baihua She should not be less than 19.3 IU·mg-1 as one of the quality control criteria of Jinlong Capsule. The study can provide a reference for the quality control of Chinese patent medicines containing animal-derived crude drugs.

Analysis of pathogenic characteristics of enteroaggregative Escherichia coli in children with diarrhea in Shanghai from 2019 to 2021 / 上海预防医学

ObjectiveTo understand the presence of virulence genes， molecular typing characteristics， and antibiotic sensitivity of enteroaggregative Escherichia coli （EAEC） in children with diarrhea in Shanghai， so as to provide a scientific basis for EAEC monitoring and standardized treatment of EAEC infection. MethodsEAEC strains isolated from children （≤5 years old） with diarrhea in six districts of Shanghai were collected as the study subjects. EAEC virulence genes were detected by real-time fluorescence quantitative PCR， molecular typing was performed by pulsed-field gel electrophoresis （PFGE）， and drug susceptibility tests were conducted using the microbroth dilution method. χ2 test and two independent samples t-test were used to compare the differences in virulence genes and antibiotic resistance between suburban and urban EAEC strains. ResultsFrom 2019 to 2021， the overall detection rates of gene aggR， pic and astA of 59 EAEC were 30.5%， 50.8%， and 57.6%， respectively. There was no significant difference in the detection rates of virulence genes between suburban and urban EAEC strains （P>0.05）. PFGE analysis revealed that only two EAEC strains belonged to the same PFGE pattern and were collected from the same hospital， and the overall PFGE patterns were polymorphic. EAEC showed susceptibility to imipenem and colistin E， and the resistance rates to sulfamethoxazole （SXT）， ampicillin （AMP）， nalidixic acid （NAL）， and tetracycline （93.1%， 79.3%， 63.8%， and 58.6%， respectively） were higher than 50.0%. The antibiotic resistance rates of cefazolin （CFZ）， cefotaxime （CTX）， and ciprofloxacin （CIP） were significantly different between EAEC strains from suburban and urban areas （P<0.05）. A total of 47 strains exhibited multi-drug resistance， with the most common resistance spectrum being AMP-SXT-NAL. There was no statistically significant difference in the number of multidrug-resistant EAEC strains between suburban and urban areas （P>0.05）. ConclusionThe EAEC virulence gene assemblages in children with diarrhea in the six districts of Shanghai are diverse， and the molecular typing patterns are relatively scattered， indicating possible cross-infection of homologous strains. Multi-drug resistance in EAEC strains is relatively common， and there is a statistically significant difference in the resistance rates of CFZ， CTX and CIP between urban and suburban EAEC strains. Attention should be given to standardizing the use of clinical antibiotics to effectively control the dissemination of multidrug-resistant EAEC strains.

Application evaluation of a rapid fluorescence quantitative PCR method for the detection of SARS-CoV-2 / 中华微生物学和免疫学杂志

Objective:To establish and evaluate a rapid nucleic acid detection method for SARS-CoV-2 based on COYOTE ? Flash20 real-time fluorescent quantitative PCR instrument. Methods:A rapid reaction system was constructed by using specific primer and probe sets targeting ORF1ab and N gene of SARS-CoV-2, and the sensitivity and specificity of the system were verified. At the same time, 108 clinical samples of COVID-19 were used to evaluate the application of this method.Results:The detection method did not require nucleic acid extraction, and the manual operation time was only one minute. After the sample was sent to the system, the test could be completed in 30 minutes. The detection limit of this method was 4×10 2 copies/ml. It had no cross-reactivity with other human coronaviruses (including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV) and other respiratory viruses. The evaluation of clinical sample application showed that the total coincidence rate with the conventional RT-qPCR which required nucleic acid extraction was 98.15%. Conclusions:Through the application evaluation of the rapid fluorescent quantitative PCR method of SARS-CoV-2, it was found that the method was simple, fast, specific and sensitive, and it was suitable for real-time and rapid detection needs in varieties of situations.

Study on the effect of chlorine disinfectants on the destruction of New coronavirus nucleic acid / 公共卫生与预防医学

Objective To observe the destructive effects of chlorine disinfectants on the nucleic acid of novel coronavirus, and to assess the feasibility of real-time fluorescent quantitative PCR technology for the evaluation of the disinfection effect of novel coronavirus. Methods A suspension quantitative inactivation test was used to observe the inactivation effects of different concentrations of chlorine containing disinfectants and different action times on the novel coronavirus. RT-PCR method was used to detect novel coronavirus nucleic acid for the disinfection effect evaluation. Results The chlorine disinfectants of 1 000 mg/L and 2 000 mg/L could destroy the nucleic acid of ew coronavirusafter application for over 30s. The chlorine disinfectant of 750 mg/L could destroy the nucleic acid of new coronavirusafter application for over 15min. Chlorine disinfectant of 500 mg/L could not completely destroy the nucleic acid of new coronavirusafter 30 min action. Conclusion The disinfection method stipulated in the national prevention and control plan using 1 000mg / L effective chlorine for the epidemic sites of new coronavirus for 30 minutes could completely destroy the nucleic acid of new coronavirus. The Real-time quantitative PCR can be used to evaluate the disinfection effect of new coronavirus.

Effect of emodin on gut microbiota of rats with acute kidney failure / 中国中药杂志

The aim of this paper was to investigate the effect of emodin on gut microbiota in acute kidney injury rats( AKI). Rats were randomly divided into several groups: normal group,model group,low-dose of emodin group( 10 mg·kg~(-1)),medium-dose of emodin group( 25 mg·kg~(-1)),high-dose of emodin group( 50 mg·kg~(-1)) and control group( 5 mg·kg~(-1) of benazepril hydrochloride).The AKI model rats were established by intraperitoneal injection of small dose of gentamicin sulfate for 7 days. Two hours after intraperitoneal injection,except for the normal group and the model group,the other groups were given corresponding doses of drugs for 15 days. The serum levels of serum creatinine( SCr),urea nitrogen( BUN),plasma endotoxin level,24 h urinary protein and D-lactate in the plasma were determined by sarcosine oxidase,urease method,tal reagent method,bromo cresol chloroform method and double antibody sandwich enzyme-linked immunoadsorbent assay,respectively. Gut microbial communities were assayed by fluorescent quantitative PCR methods. HE staining was used to detect the pathological changes of the kidneys. Compared with the normal group,there were significant differences in body weight,urinary protein( UTP),bacterial endotoxin,urea nitrogen,creatinine,D-lactate in the plasma and four bacterial contents in the model group( P<0. 05). The urinary protein,urea nitrogen,D-lactate,creatinine and plasma bacterial endotoxin in control group and each emodin group were lower than those in model group,especially for high-dose of emodin( P<0. 01). Moreover,pathology resolution in high-dose emodin was better than other groups. Except for low-dose of emodin group,qRT-PCR data suggested that the amounts of Escherichia coli and Enterococcus in medication administration group were increased,while the amounts of Lactobacilli and Bifidobacterium were reduced compared with model group( P<0. 05),especially for high-dose of emodin( P<0. 01). There is a clear imbalance of gut microbiota in rats with AKI. Emodin could regulate the imbalance of gut microbiota,which might be one of the mechanisms of its effects on AKI rats.

Identification and expression pattern analysis of a Moso Bamboo LTR retrotransposon / 生物工程学报

To develop more active LTR retrotransposons in Phyllostachys edulis, a Ph. edulis LTR retrotransposon (Ph-LTR2) was identified, and the expression pattern of the transposon under stress was systematically analyzed. Ph-LTR2 transposon is 6 030 bp in length and belongs to the Reina subfamily in the Ty3-Gypsy family. With the similarity of 96.41% of both LTR sequences, the Ph-LTR2 transposon inserted the moso bamboo genome about 61.92 thousand years ago. There are 5 copies identified in the genome. The Ph-LTR2 transposon domain includes GAG (gag protein) protein domain, PR (Proteases) protein domain, RT (Reverse transcriptase) protein domain, RH (Ribonuclease H) protein domain, INT (Integrase) protein domain and CHR (Chromatin organization modifier) protein domain. The expression patterns of INT, RT and RH were detected by real-time quantitative PCR. The three domains were found to have specific expression patterns at different tissues of the bamboo. Under the conditions of low/high temperature, methylation inhibitors treatments, irradiation and high salt stress, transcription levels of the three domains of the Ph-LTR2 transposon increased with different degrees. Specifically, after treatment with low/high temperature and methylation inhibitors, the transcription level was up-regulated; after low dose radiation treatment and low concentration of salt solution treatment, the transcription level was also increased, but the expression level decreased with increasing dose of radiation and concentration of salt solution. These results indicate that the expression pattern of the Ph-LTR2 transposon responds to the changes of the external environment, but the exact mechanism is not yet known. The results of this study laid a certain theoretical foundation for the development of the genetic tool based on Ph-LTR2 transposons.

Establishment and verification of real-time fluorescent quantitative PCR detection system for ring virus 6 / 中华实验和临床病毒学杂志

Objective@#To establish a real-time quantitative PCR detection system for Torque teno virus (TTV) and verify the sensitivity and specificity of the detection system.@*Methods@#Primers and FAM-Eclipse probes were designed based on the TTV6 gene sequence registered in GenBank, and were to establish a real-time fluorescent quantitative PCR detecting way based on the FAM-Eclipse probe, the standard curve was constructed and sensitivity and specificity were analyzed.@*Results@#A quantitative PCR method for the specific detection of TTV6 were established that the standard curve equation was y=-3.0921x + 28.36, and the amplification efficiency and R2 were 99.6% and 1.000, respectively. The sensitivity of TTV6 was 1.0×10 copies/μl, and there was no cross-reactivity with other viruses. There was 1 case positive for TTV6 out of 56 throat swab samples from the patients with clinical respiratory infection.@*Conclusions@#The real-time fluorescent quantitative PCR for detecting TTV6 established by FAM-Eclipse probe had the advantages of high sensitivity and specificity. It provides an effective way for detection and quantification of viral content of TTV6 in clinical specimens.

Pathogen spectrum of viral encephalitis in children living in Hebei province, China from May to December 2017 / 中华实验和临床病毒学杂志

Objective@#To investigate the pathogenic characteristics of viral encephalitis in children living in Hebei province.@*Methods@#We randomly collected cerebrospinal fluid specimens from a total of 399 children diagnosed with viral encephalitis in Hebei Children′s Hospital from May to December 2017. Real-time fluorescence quantitative PCR and Sanger sequencing were used to detect viral nucleic acids in cerebrospinal fluid by an automatic laboratory station. Statistical analysis was performed on the experimental data using SPSS 21.0 software and the clinical data were analyzed. Comparison of infection rates of EV encephalitis in different months, using line × column chi-square test. The MRI and EEG positive rates of different viral encephalitis and viral encephalitis patients not infected with the virus were analyzed by Fisher′s exact probability test. The positive rate of infection with different viruses and non-virus agents was analyzed by Fisher′s exact probability test.@*Results@#The result showed that 80 of 399 samples were positive, and the positive rate was 20.05%. It included 22 cases of enterovirus, 4 cases of influenza A virus, 3 cases of mumps virus, 2 cases of herpes simplex virus type 1, 1 case of herpes simplex virus type 2, 4 cases of EB virus, 7 cases of cytomegalovirus, 7 cases of herpes zoster virus, 8 cases of adenovirus, 14 cases of human herpesvirus type 6. Eight cases had combined viral infection. Eight cases had concurrent infections: 3 cases had enterovirus and herpesvirus type 6 concurrent infection, 1 case had enterovirus and Japanese encephalitis virus concurrent infection and 1 case had herpes simplex virus type 2 and adenovirus, 1 case had influenza A virus herpesvirus type 6, 1 case had mumps virus and herpesvirus type 6, 1 case had mumps virus and herpesvirus type 6, 1 case had herpes simplex virus type 1 and herpes zoster virus concurrent infections. Children with EV viral encephalitis in Hebei Province were highly prevalent in May and June (P=0.016). HHV6 virus encephalitis was more susceptible to infection than non-HHV6 virus (P=0.016); The rate of MRI positive findings in patients with different viral encephalitis was not statistically significant (P>0.05). The result of EEG of different viral encephalitis were P>0.05, which was not statistically significant.@*Conclusions@#EV was the most common pathogen of children with viral encephalitis in Hebei province. Encephalitis caused by influenza A virus cannot be ignored in clinical practice.

Imported Plasmodium ovale malaria in Hefei City: a case report / 中国血吸虫病防治杂志

Objective To perform epidemiological survey and laboratory diagnosis of a Plasmodium ovale malaria case imported into Hefei City, so as to provide scientific evidence for the prevention and control of imported malaria in the future. Methods The epidemiological history and clinical data of the imported malaria case were collected and analyzed. Results The patient returned to China from Mozambique, and was admitted to the hospital due to repeated fever several months after returning to China. Rapid diagnostic test (RDT) suggested non-P. falciparum infection, and microscopy displayed normal or slightly swelled malaria parasite-infected erythrocytes, unapparent serrated changes, increased cytoplasm in large trophozoite with irregular morphology, and almost no vacuoles. qPCR assay revealed P. ovale infection. Conclusions Clinical medical professionals should improve their awareness of malaria diagnosis in people with a history of living or working in overseas malaria-epidemic areas, and early and rational administration of antimalarials should be given promptly to prevent the spread of malaria epidemics.

Imported Plasmodium ovale malaria in Hefei City: a case report / 中国血吸虫病防治杂志

Objective To perform epidemiological survey and laboratory diagnosis of a Plasmodium ovale malaria case imported into Hefei City, so as to provide scientific evidence for the prevention and control of imported malaria in the future. Methods The epidemiological history and clinical data of the imported malaria case were collected and analyzed. Results The patient returned to China from Mozambique, and was admitted to the hospital due to repeated fever several months after returning to China. Rapid diagnostic test (RDT) suggested non-P. falciparum infection, and microscopy displayed normal or slightly swelled malaria parasite-infected erythrocytes, unapparent serrated changes, increased cytoplasm in large trophozoite with irregular morphology, and almost no vacuoles. qPCR assay revealed P. ovale infection. Conclusions Clinical medical professionals should improve their awareness of malaria diagnosis in people with a history of living or working in overseas malaria-epidemic areas, and early and rational administration of antimalarials should be given promptly to prevent the spread of malaria epidemics.

Differential pathway and gene expressions of endophytic Phialocephala fortinii inoculated on Rhodiola crenulata / 药学学报

The development and metabolism of medicinal plant is affected by many factors, among which the effect from endophytic fungi has been noticed recently and has become one of hot fields. In order to explore the effect of endophytic fungi on gene expression in R. crenulata, RNA-sequencing was used to find genes involved in metabolic pathways, and the differential genes were verified by real-time fluorescent quantitative PCR. The method of 2-△△Ct was used to analyze the relative expression levels of genes in related metabolic pathways, which was used to verify the result of transcriptomics sequencing. The results showed that the endophytic fungus, P. fortinii, could up-regulate the gene expression in lipid metabolic pathway of R. crenulata. In signal transduction pathway, the genes were influenced at different level but the gene expressions were significantly increased under control of Notch signaling pathway, which was 34 times of that in control. The gene expressions of environmental adaption pathway were up-regulated in R. crenulata after inoculation of P. fortinii. This study could provide help for further understanding on mechanism of plant-fungus interaction, root cause of geoherbalism of medicinal plant and exploring bio-function of endophytic fungi.

Comparison and evaluation of laboratory test techniques for severe fever with thrombocytopenia syndrome / 中华实验和临床病毒学杂志

Objective@#Compare the detection result of blood samples of severe fever with thrombocytopenia syndrome (SFTS) patients using different detection techniques, and observe the dynamic characteristics of the virus specific RNA, IgM antibody and IgG antibody, to provide theoretical basis for selection of diagnostic methods of disease.@*Methods@#Acute phase serum of suspected SFTS cases and convalescent serum samples of lab-confirmed cases were collected. Real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the virus specific RNA, IgM antibody and IgG antibody. The detection results of different methods, the relationship between positive results and the acquisition time, and the dynamic characteristics of viral nucleic acid and antibodies were analyzed.@*Results@#A total of 87 serum samples of the suspected SFTS patients were collected, the positive rate of virus specific RNA, IgM antibody and IgG antibody were 53.41%, 31.03% and 3.41%, respectively. Among 55 confirmed cases of SFTS, the consistent rate of virus specific RNA and IgM antibody detection methods was 36.36%, and the difference between the two methods was significant (χ2=6.82, P=0.009), kappa=-0.257. The sampling intervals of RNA positive samples were all within 12 days, of which the positive detection rate was highest after 7-9 days, and the difference was statistically significant (χ2=10.35, P=0.016). In 34 SFTS convalescent serum samples, all the nucleic acid tests were negative, the positive rate of IgM antibody was 41.18%, which was not significantly different from the acute phase serum samples (P=1.00). The positive rate of IgG antibody was 94.12%, which was significantly higher than that of acute IgG antibody (0%). The dynamic characteristics of IgM and IgG antibody showed that IgM antibody could be detected on the second day after onset, the latest detection time was 74 days after onset, and the highest absorbance value and antibody detection rate occurred in 30-60 days. The earliest detection time of IgG antibody was 12 days after onset, and the last detection time was 100 days.The detection rate of IgG antibody and absorbance value increased rapidly after 30 days, and maintained in a high level. The detection rate of IgG antibody was 100% in 30-60 days.@*Conclusions@#Blood samples taken from SFTS suspected patients within two weeks of onset may be prioritized for detection of viral nucleic acids using Real-time fluorescence PCR or for detection of IgM antibodies by ELISA. Although IgM antibody can be detected 2 days after the onset, the peak appeared much later, so the negative result can’t rule out the diagnosis. IgG antibody has a high seroconversion rate in convalescent samples, and can be used as an auxiliary tool for disease diagnosis.

Expression of FFAR4 protein and mRNA in pancreatic cancer and its clinical significance / 临床与实验病理学杂志

Purpose To detect the expression of FFAR4 protein and mRNA in pancreatic cancer and to discuss its role and significance in the progression of pancreatic cancer. Meth-ods Immunohistochemistry was used to detect the expression of FFAR4 protein in paraffin-embedded tissues from 62 cases of pathologically confirmed pancreatic cancer. The relationship be-tween the expression of FFAR4 and clinicopathological factors of pancreatic cancer was also studied. At the same time, the ex- pression of FFAR4 in 20 pairs of pancreatic cancer tissues and adjacent tissues were detected using Western bolt and real-time quantitative PCR (qRT-PCR). Results The results of immu-nohistochemistry showed that the expression rate of FFAR4 pro-tein in pancreatic carcinoma was 75. 8% (47/62) significantly higher than that in paratumor tissue 40. 3% (25/62), and the difference was statistically significant (P＜0. 05). The high ex-pression of FFAR4 was related to the degree of pancreatic cancer differentiation, TNM stage, and lymph node metastasis ( P ＜0. 05). Western blot and qRT-PCR showed that the expression of FFAR4 protein and its mRNA expression in pancreatic cancer tissues was significantly higher than matched paracancerous tis-sues. There was a statistically significant difference between the two groups ( P ＜0. 001 ). Conclusion The dysregulated ex-pression of FFAR4 may be closely related to the progression of pancreatic cancer. It is hopeful that FFAR4 may become a new marker for the prognosis of pancreatic cancer after surgery and a new target for the study of clinical therapeutic drugs.

Expression of c-IAPl protein and mRNA in gastric adenocarcinoma and its clinical significance / 临床与实验病理学杂志

Purpose To investigate the protein and mRNA expression of c-IAPl in gastric adenocarcinoma tissue and its clinical significance. Methods Immunohistochemistry technique, Western blot and realtime fluorescent quantitative PCR(qRT-PCR) were used to detect the protein and mRNA expression of c-IAPl in 50 cases of gastric adenocarcinoma tissues and40 cases of adjacent normal gastric mucosa tissues to analyze its role in the development and progression of gastric adenocarcinoma, the relationship between the protein and mRNA expression of c-IAPl and the clinicopathological features. Results The relative expression level of c-IAPl protein in gastric adenocarcinoma tissues was significantly higher than that in adjacent normal gastric mucosa tissues, the difference was statistically significant (P＜ 0.05 ). The mRNA expression of c-IAPl in gastric adenocarcinoma was significantly higher than normal gastric mucosa tissues, the difference was statistically significant (P＜0.05). The protein and mRNA expression of c-IAPl were correlated with the degree of differentiation of gastric adenocarcinoma tissues, TNM clinical stage, lymph node metastasis and infiltration depth, the difference was statistically significant (P＜0.05 ), while there was no correlation with gender and age, the difference was not statistically significant (P＞ 0.05). Conclusion The high protein and mRNA expression of c-IAPl in gastric adenocarcinoma tissues inhibit the apoptosis of gastric adenocarcinoma cells, which contribute to the development and progression of gastric carcinoma and it may provide a new theoretical basis for the clinical targeted therapy of gastric adenocarcinoma.

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 .

Effect of endophytic fungus on expression of key enzyme genes in pathway of salidroside biosynthesis in Rhodiola crenulata / 药学学报

One strain of endophytic fungus ZPRa-R-1 was obtained for the capacity of promoting production of salidroside in Rhodiola crenulata. To explain the mechanism of salidroside biosynthesis in host plant, eight housekeeping genes were evaluated, and the evaluation method was created for the expression activities of four key enzyme genes PAL (phenylalanine ammonia-lyase), TyDC (tyrosine decarboxylase), TAT (tyrosine transaminase), UDPGT (UDP-glucosyltransferase) referenced double reference genes in biosynthesis pathway of salidroside in R. crenulata. Stabilities of housekeeping genes were confirmed by real-time fluorescent quantitative PCR technology and three softwares including geNorm, NormFinder and BestKeeper, then relative expressions of key enzyme genes were analysized by the 2-ΔΔCt method. The results showed that the most stable gene was GAPDH, followed by PCS, and the most appropriate reference of internal genes were combination with two genes in R. crenulata inoculated with endophytic fungus ZPRa-R-1. Under symbiosis conditions, regularity changes of key enzyme genes affected by endophytic fungus ZPRa-R-1 were as follows:the relative expression activity of PAL attached to peak value, which was 4.9 times as that of control group when inoculated ten days. The relative expression of TyDC reached the maximum value, which was 2.8 times of that control after inoculating 12 days. The relative expression of UDPGT actually reach 17.1 times than that of control after inoculating 8 days. However, the relative expression of TAT was not affected by this fungus. The changes of four key enzyme genes are positively correlated with the changes of salidroside content in R. crenulata.

Establishment and preliminary application of a real-time fluorescent quantitative PCR assay for detection of murine norovirus / 中国比较医学杂志

Objective To establish a real-time fluorescent quantitative PCR ( FQ-PCR ) method for detection of murine norovirus ( MNV) in laboratory mouse and provide the basis for establishment of a standard detection method for MNV.Methods Specific primers were designed and MNV DNA standards were prepared according to the MNV genome sequences published on NCBI .The specificity, sensitivity, repeatability and stability of the established Q-PCR method were tested.The established Q-PCR method was applied to detect 766 mouse caecum content samples to explore preliminarily the infection status of laboratory mice in Beijing .Results No cross reaction showed in human norovirus and feline calicivirus with the established Q-PCR method.The sensitivity was up to 10 copies/μL.The coefficient of variation ( CV) of intra-assay and inter-assay was less than 2%.There were 301 positive cases detected in the 766 samples of laboratory mice.Conclusions The established FQ-PCR method is accurate and effective with high specificity , sensitivity and repeatabiliy in the quantitative detetion of nucleic acid , and can be applied to rapidly and quantitatively screen MNV in laboratory mice .

Effects of curcumin on gut microbiota of interval sleep deprivation rats / 中草药

Objective: To study the effects of curcumin (CUR) on gut microbiota of interval sleep deprivation (ISD) rats. Methods: Wistar rats were randomly divided into control group, big platform (BP) control group, model group, and CUR (70 mg/kg) group. Firstly, ISD model rats were established by improved small platform method; Then, open field test and sucrose preference test were utilized to assess the depression-like behavior of the ISD model rats and intervention effects of CUR; Thirdly, gut microbiota DNA samples were extracted from feces, the primers of Escherichia coli, Bifidobacterium, Lactobacillus, Clostridium perfringens, and Bacteroides were designed by 16S rRNA gene sequence, and the fluprescence qualitation of the bacteria through general PCR was obtained. Results: The results of behavioral experiments showed that open field test scores and sucrose preference rate were significantly lower than those of the BP group (P<0.01), and CUR played a significant improving role. The qRT-PCR data suggested that the relative expression of E. coli, Bifidobacterium, Lactobacillus, and Bacteroides of ISD model rats were lower than those in normal and big platform groups in ISD model rats, while higher for Clostridium perfringens (P<0.05, 0.01). The amounts of E. coli, Bifidobacterium, Lactobacillus, and Bacteroides of CUR intervened group were higher than those in ISD model rats, except for Clostridium perfringens (P<0.05, 0.01). Conclusion: The results show that as a stress, ISD not only causes mental disorders in rats, but also leads to the changes of five bacteria strains. CUR could alter the imbalance of gut microbiota, which might be one of the mechanisms of its effects on depression-like behaviors of ISD rats.