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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Genome , Phylogeny , Poaceae , RetroelementsABSTRACT
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.
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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 .
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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.
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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.
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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.
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OBJECTIVES@#To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval （PMI）.@*METHODS@#Twelve individuals with known PMI （range from 4.3 to 22.5 h） were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including β-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI.@*RESULTS@#5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using β-actin was 24.6%, while GAPDH was 41.0%.@*CONCLUSIONS@#5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of β-actin correlates well with PMI, which can be used as an additional index for early PMI estimation.
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Humans , Actins/analysis , Autopsy , Brain/metabolism , MicroRNAs/analysis , Models, Theoretical , Postmortem Changes , RNA Stability , RNA, Ribosomal, 18S/analysis , RNA, Ribosomal, 5S/analysis , RNA, Small Nuclear/analysis , Real-Time Polymerase Chain Reaction , SoftwareABSTRACT
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.
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Objective: To clone and analyze the coding region of gibberellin 20-oxidase (GA20ox) gene of Panax quinquefolium in seed dormancy and germination process. Methods: According to P. quinquefolium seeds transcriptome annotation, one transcript coding of GA20ox was obtained by comparative analysis of nine related unigenes. The full-length cDNA of PqGA20ox was determined by using RT-PCR method. Then the bioinformatic analysis of this gene and encoded protein was performed. The expression level of PqGA20ox in the roots, stems, leaves, and several seed dormancy periods was detected by real time fluorescent quantitative PCR (RT-qPCR). Results: Bioinformatic analysis showed that PqGA20ox contained 1092 bp and encoded 363 amino acids. PqGA20ox encoded protein had neither transmembrane nor signal peptide. The expression level of PqGA20ox was higher in the inset periods of morphological and physiological dormancy than that in the other periods based on RT-qPCR analysis. Conclusion: The PqGA20ox gene is cloned for the first time, and will provide a foundation for the dormancy release molecular mechanism of P. quinquefolium.
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Objective To investigate the expression of CD44 varant 2(CD44v2) in bladder and urothelial carcinoma ,and to study its significance in the diagnosis of human bladder and urothelial carcinoma .Methods Real‐time fluorescent quantitative PCR was used to analyze the expression of CD44v2 protein in 70 bladder and urothelial carcinoma tissue samples from patients in different pathological and clinical stages .Meanwhile ,20 tissue samples of normal bladder mucosa were collected as controls .Results CD44v2 expression was negative in normal bladder and urothelial mucosa ,the gene copies were less than 1 × 102 copies/mL ,while the posi‐tive expression rate of CD44v2 was 42 .9% (30/70) ,and Ct values were less than 35 and copy number was greater than 1 × 104 cop‐ies/mL .Positive expression rate was correlated with high pathological grades and TNM stages .Conclusion CD44v2 could be an useful indicator for the early assessment of bladder and urothelial carcinoma .
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Objective To establish the method of the SYBR Green Ⅰ real‐time fluorescent quantitative PCR for detecting the se‐rum miR‐203 expression level ,and to detect the serum miR‐203 expression levels in the patients with cervical cancer ,cervical benign diseases and healthy controls .Methods The miR‐203 ,U6 stem loop RT primers and the PCR amplification primers were designed for conducting fluorescence quantitative PCR ,with U6 as the internal relative quantification ,the serum miR‐203 levels were com‐pared among different cervical diseases .Results The established method could specifically detect the amplification signal of serum miR‐203 ,the melting curve was single and PCR products were specific .The serum miR‐203 level in the patients with cervical cancer was significantly higher than that in the patients with benign cervical diseases such as hysteromyoma and cervicitis ,the difference was statistically significant (P<0 .05) .Conclusion The SYBR Green Ⅰ real‐time fluorescent quantitative PCR is a quick ,simple detection method with high sensitivity and good specificity ,which may have a better application prospect in cervical cancer auxiliary diagnosis .
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Objective To participate in the recognition of medical laboratory and to evaluate the performance of the instruction, we assess the performance of ABI 7300 real-time fluorescent quantitative PCR instrument.Methods According to United States of America Clinical and Laboratory Standards Institute(CLSI),assess the precision,accuracy,sensitivity and linearity of HBV-DNA detection result in ABI 7300 fluorescence quantitative PCR instrument.Results The Intraassay coefficient of variation is 2.38%-4.61%,and interassay coefficient of variation was 3.87%-5.33%,both achieve the performance analysis standard of gene ampli-fication test project regulated by Medical Laboratory Quality and Competence Accreditation Criteria.Accuracy:the overall mean bias in 2011 Ministry of Health EQA results was 2.14%,which met the quality assessment requirements of the ministry of health. Functional sensitivity:the CV of 100 IU/mL detection limit concentration was close to 20%,which accords with the The limit of detection of the kit;Linear:the correlation coefficient r2 is 1.0,linear relationship met the requirements.Conclusion The perform-ance index of ABI 7300 real-time fluorescent quantitative PCR instrument is accuracy,and it can provide fast,accurate reports for clinical department.
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Objective To explore the difference in bacterial flora between faeces and mucosa of sigmoid colon,the possible role and significance of microbiota alteration in the genesis of ulcerative colitis (UC).Methods Fusobacterium, Enterococcus, Lactobacillus, Bifidobacterium, Bacteroides and Escherichia were selected as target bacteria colonies.The content of six target bacteria colonies in faeces and mucosa of sigmoid colon of 35 UC patients (20 active UC, and 15 UC in remission) and 20 health controls were detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR).Two independent samples t-test was performed to compare the differences in bacterial flora between faeces and mucosa of sigmoid colon.Variance analysis was used to compare the differences in bacterial flora among health controls group,active stage group and remission stage group.Results In health control group, the contents of Fusobacterium, Bacteroides, Enterococcus and Lactobacillus in faeces ((10.94 ± 0.29),(12.42±0.39), (8.73±0.84), (9.05±0.35), respectively) were higher than those in the mucosa of sigmoid colon ((9.36±0.66), (9.88±0.82), (7.70±1.17) and (7.96±0.68), respectively, t=9.83, 12.51, 3.20 and 6.35, all P＜0.05).However, the content of Escherichia was lower in faeces than that in the mucosa of sigmoid colon ((8.03±1.02) lg copy/g vs (8.91±0.52) lg copy/g, t=-3.44, P＜0.05).There was no difference in the content of Bifidobacterium between faeces and mucosa of sigmoid colon ((9.54±0.79) lg copy/g vs (9.42±0.98) lg copy/g, P＞0.05).For UC patients, the contents of Fusobacterium, Bacteroides, Enterococcus, Lactobacillus and Bifidobacterium in faeces ((9.62 ±± 1.13),(11.31±0.71), (9.33±0.65), (8.42±0.80) and (8.85±0.73) lg copy/g, respectively) were higher than those in the mucosa of sigmoid colon ((9.00±0.79), (8.80±0.66), (7.46±0.82), (6.82±1.07) and (8.40±0.72) lg copy/g, respectively, t=2.66, 15.28, 10.58, 7.12 and 2.56, all P＜0.05).The content of Escherichia was lower in faeces than that in the mucosa of sigmoid colon ((8.50 ± 0.52) lg copy/g vs (9.26±0.87) lg copy/g, t=-4.45, P＜0.05).Compared with health control group, the content of Fusobacterium, Lactobacillus, Bifidobacterium and Bacteroides ((8.83 ± 0.81), (7.48 ± 1.59), (8.55±0.79) and (11.11±0.88) lg copy/g) all decreased (F=84.45, 14.58, 10.43 and 24.91, all P＜0.05), while the contents of Enterococcus and Escherichia increased ((9.63 ± 0.39) and (8.74 ±0.53) lg copy/g, F=9.87 and 5.55,both P＜0.05).For remission stage group, only the content of Bacteroides decreased ((11.56±0.21) lg copy/g, P＜0.05).Compared with health control group, the contents of Fusobacterium, Bacteroides, Lactobacillus, Bifidobacterium ((8.52 ± 0.30), (8.34 ±0.29), (6.29±0.52) and (8.06±0.21) lg copy/g) all decreased in active stage group (F=16.99,35.98,22.28 and 16.08, all P＜0.05);the content of Escherichia increased ((9.68±0.56) lg copy/g, F=11.19,P＜0.05);there was no difference in the content of Enterococcus ((7.19±0.32) lg copy/g, P＞0.05).In remission stage group, the contents of Bacteroides fragilis and Bifidobacterium decreased ((9.42±0.48) lg copy/g and (8.87±0.89) lg copy/g, both P＜0.05), there was no difference in other bacterias (all P＞0.05).In both faeces and mucosa of sigmoid colon, the ratios of Bifidobacterium and Enterobacteriaceae (B/E value) in active stage group were less than 1 (0.98±0.13 and 0.84±0.05),which significantly decreased compared with health control group (1.21 ± 0.19, 1.06 ± 0.08;F=12.64,76.20, both P＜0.05).In remission stage group, B/E values were higher than 1 both in faeces and mucosa (1.14±0.08 and 1.02±0.04), and there was no difference compared with those of control group (P＞0.05).Conclusions The distribution of target bacteria in feces and sigmoid colonis differed between health controls and UC patients.There are obvious changes in fecal and mucosa associated bacterial flora in UC patients especially in active stage compared with healthy controls.
ABSTRACT
Objective To establish and evaluate a universal real-time fluorescent quantitative PCR(qPCR)method for identifying and quantifying three human parvovirus B 19 ( B19V) genotypes.Methods Firstly, following a bioinformatic analysis of a subset of B19V genomic sequences available in the NCBI nucleotide database ,representative of genotypes 1 to 3,a set of suitable universal primers and TaqMan probes was designed from the NS 1 gene of B19V.Aplasmid was used as a quantitative standard that contained the identical sequence of the B 19 target sequence .An internal control ( IC ) was included to prevent false negative results .Then,serial 1-log dilutions of quantitative standards were prepared and used in the qPCR assays for generation of a standard curve .Finally,the specificity,sensitivity and reproducibility of the assay were assessed.Results A linear relationship of the real-time PCR method for detecting B19V from 1 ×109copies/μl to 1 ×103 copies/μl was observed .The developed qPCR protocols allowed for the detection of genotypes 1 to 3 with a limit of detection ( LOD) of 10 copies/μl.Furthermore, the assay did not amplify other blood-borne viruses.The inter-and intra-assay variability analyses showed good reproducibility of the assay .Conclusion A universal real-time qPCR method for the detection of B19V DNA is established,which will facilitate the diagnosis of B19V infections and the screening of blood and plasma-derived products , thereby improving the viral safety of transfusion and plasma-derived products .