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A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.
Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.
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Humans , Male , Female , Adult , Middle AgedABSTRACT
SUMMARY OBJECTIVE: The aim of this study was to assess the results and efficiency of two real-time polymerase chain reaction procedures for detecting human papillomavirus utilizing urine samples. METHODS: This study comprised 151 patients who had previously tested positive for human papillomavirus in their cervical samples. Two different commercial real-time polymerase chain reaction techniques were used for identification and genotyping human papillomavirus in urine specimens. The urine samples of 151 patients were evaluated via the Roche Cobas test, and the urine samples of 91 patients were also evaluated via the Qiagen test. RESULTS: The overall consistency of urine and cervical swab specimens for the identification of human papillomavirus in Roche Cobas and Qiagen tests were 44.8 and 44%, respectively. The rates of positive human papillomavirus results from urine samples were 57 and 70.3%, respectively. The overall concordance among Roche Cobas and Qiagen tests utilizing urine samples for human papillomavirus type 16/18 was 84.3% with a kappa value of 0.675, and for other high-risk-human papillomavirus, it was 75.60% with a kappa value of 0.535. Roche Cobas showed high concordance with Qiagen test. CONCLUSION: human papillomavirus positivity was not detected in all urine samples. It is still inappropriate to recommend the use of urine liquid biopsy for the accurate and reliable detection of human papillomavirus. Due to the lack of a standardized tool, the utilization of urine samples as a screening human papillomavirus test remains a challenge.
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ABSTRACT Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.
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Objectifs.Evaluer l'apport de la TDM thoracique dans le diagnostic de la COVID-19 chez les patients suspects en prenant comme référence le Test RT PCR et secondairement déterminer les anomalies scanographiques caractéristiques associés au Test RT PCR.Matériels etméthodes.Il s'est agi d'une étude rétrospective, descriptive à visée analytique, menée au service d'imagerie du CHU de Libreville. Aux vues de la disponibilité des dossiers nous avons exploité ceux de la période d'activité de mars à juin 2020, soit une période quatre mois. Cette étude a inclus tous les patients ayant une infection à COVID-19 confirmée par la réaction de polymérase en chaine en temps réel (RT PCR), après écouvillonnages nasal et/ou pharyngé et / ou les signes typiques de la pneumonie à COVID-19. Pour déterminer les anomalies scanographiques caractéristiques de la COVID-19 associées au test RT PCR nous avons comparé 25 patients avec un test RT PCR négatifs et 119 patients avec un test RT PCR positifs. Une valeur de p < 0,05 était considérée comme significative. La sensibilité de la TDM thoracique a été calculée à l'aide de la relation, Sensibilité=VP/ (VP+FN), (VP : vrai positif de la TDM : TDM positive et test RT PCR positif ; et le FN : Faux négatif de la TDM : TDM négative et test RT PCR positif). L'analyse statistique a été rendu possible grâce au logiciel SPSS 25. Résultats. En régression logistique multivariée, les anomalies scanographiques caractéristiques de la COVID-19 positivement associés à un test RT PCR positif étaient :verre dépoliOR = 33,236, IC95% [6,939 59,192], p=0,0001), la condensation alvéolaire (OR = 8,093 [1,488 44,013]; p=0,016). La sensibilité du scanner thoracique pour le diagnostic de la COVID-19, chez les patients suspects était de 93,2%, la spécificité de 64,2% avec une valeur prédictive positive de 90,2% et une valeur prédictive négative de 73,0%.Conclusion.Le diagnostic scanographique de la COVID-19 chez les patients suspects est aisé avec une bonne sensibilité et spécificité. La présence des anomalies scanographiques caractéristiques à type de verre dépoli et de condensation alvéolaire est associéeà un test RT PCR positif
Objective. Evaluate the contribution of Thoracic CT in the diagnosis of COVID-19 in suspected patients by taking the RT PCR Test as a reference and secondarily determine the impact of the RT PCR Test on characteristic scan abnormalities, in the service of thoracic imaging at Libreville University Hospital.Materials and methods. This was a retrospective, descriptive study with an analytical aim. In view of the availability of files, we used those from the period of activity from March to June 2020, i.e. a four-month period. To determine the impact of the RT PCR test on characteristic scan abnormalities, we compared 25 patients with negative RT PCR and 119 patients with negative RT PCR. A value of p < 0.05 was considered significant. The sensitivity of chestCT was calculated using the relationship, Sensitivity=VP/(VP+FN), (VP: true positive of CT: positive CT and positive RT PCR test; and FN: False negative CT: negative CT and positive RT PCR test). Statistical analysis was made possible using SPSS 25 software.Results. In multivariate logistic regression, CT abnormalities characteristic of COVID-19 were positively associated with a positive RT PCR: ground glass, (OR = 33.236, 95% CI [6.939 59.192], p = 0.0001), condensation alveolar (OR = 8.093 [1.488 44.013]; p = 0.016). The sensitivity of chest CT for the diagnosis of COVID-19 in suspected patients was 93.2%, the specificity of 64.2% with a positive predictive value of 90.2% and a negative predictive value of 73. 0%.Conclusion. CT diagnosis of COVID-19 in suspected patients is easy with good sensitivity and specificity. The presence of characteristic scan abnormalities such as ground glass and alveolar condensation is associated with a positive RT PCR tes
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Humans , Male , Female , Sensitivity and Specificity , Academic Medical Centers , COVID-19ABSTRACT
Objective To explore the expression, correlation with clinicopathologic parameters, and clinical significance of MIS18 binding protein 1 (MIS18BP1) in bladder cancer. Methods TCGA and GEO databases were used to analyze the mRNA expression of MIS18BP1 in tumors and controls, and the results were verified via qRT-PCR. UALCAN online database was utilized in the analysis of the expression of MIS18BP1 and its correlation with clinicopathological parameters and the degree of immune cell infiltration. Immunohistochemistry was employed to analyze the expression of MIS18BP1 in bladder cancer and its relationship with clinicopathological features. The ROC curve was applied to evaluate the diagnostic value of MIS18BP1 mRNA in bladder cancer. Results Bioinformatics analysis and qRT-PCR results revealed the increased expression of MIS18BP1 mRNA in bladder cancer compared with that in the control group (P<0.05). Immunohistochemistry unveiled the significantly high positive rate of MIS18BP1 protein in bladder cancer (P<0.05) and its correlation with the clinical stage of tumors, depth of invasion, and lymph node metastasis (P<0.05). The immune infiltration analysis showed the association of MIS18BP1 with immune cell infiltration in bladder cancer. Conclusion The increased expression level of MIS18BP1 gene and protein in bladder cancer may regulate the development of bladder cancer by influencing immune cell infiltration.
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Objective To investigate the association between the index finger and ring finger length ratio (2D ∶ 4D) and of four loci (rs6461992‚ rs6968828‚ rs7801581‚ rs17427875) polymorphism of homeobox (HOX) A11 gene among Ningxia college students. Methods Digit camera was used to collect frontal hand photos of 667 Han college students (348 males and 319 females) from Ningxia province; Image analysis software was used to mark the anatomical points and measure finger lengths of the index and ring fingers of both hands; multiplex PCR was used to detect each locus polymorphisms of HOXA11 gene; statistical software was used to compare and analyze the differences and associations of 2D ∶4D and gene polymorphisms between different genders. Results Among Ningxia Han college students‚ both left hand and right hand 2D ∶ 4D were significantly higher in females than those of in males (all P< 0. 05)‚ and there were no significant sex differences in right-left hand 2D ∶4D; the genotypes and allele frequencies of rs7801581 locus of HOXA11 gene differed significantly between genders (all P < 0. 05)‚ and none of the other locus polymorphisms showed any significant sex differences; only female left hand 2D ∶4D was significantly associated with rs6461992 locus genotype in the relationship between 2D ∶4D and HOXA11 polymorphisms (P<0. 05). Conclusion There were significant sex differences in 2D ∶ 4D among Han college students in Ningxia‚ and the rs6461992 locus polymorphism of HOXA11 gene may be associated with the formation of 2D ∶4D in females.
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Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells. Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay. Different concentrations of Chir99021 were set, and the cell proliferation was detected by CCK⁃8 to select the optimal concentration. Osteogenic differentiation was detected by alizarin red staining. The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1 (Wnt1), Wnt3a and Wnt3a β⁃expression of catenin, axis inhibition protein 2(Axin 2), dentin sialophosphoprotein(OCN) and dentin matrix acidic phosphoprotein 1(DMP1) was detected by Real⁃time PCR and Western blotting. Results The positive expression of dentin sialophosphoprotein (DSPP) and vimentin indicated that rat dental pulp stem cells were successfully isolated. After osteogenic induction of rat dental pulp stem cells, calcium deposits significantly increased with the addition of glycogen synthase kinase⁃3β(GSK⁃3β) inhibitor Chir99021, calcium deposits were significanted reduced. After osteogenic differentiation of rat dental pulp stem cells, the expression of Wnt1, Wnt3a, β⁃catenin, Axin2, OCN and DMP1 increased, while the expression of Wnt1, Axin2, OCN and DMP1 decreased with the addition of Chir99021. Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.
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Objective To clarify the expression and distribution of brain⁃derived neurotrophic factor (BDNF) in the cerebrum of plateau yaks and cattle, and to explore the relationship between BDNF function and the adaptability of altitude hypoxia. Methods Five yaks and five cattles were selected.The content and distribution of BDNF in frontal lobe, temporal lobe, parietal lobe, occipital lobe, cerebrum white matter and hippocampus of yak and cattle were analyzed by Real⁃time PCR, Western blotting and Immunohistochemistry. Results Real⁃time PCR result showed that BDNF mRNA expression in the cerebrum of yaks and cattles was highest in temporal cortex, followed by hippocampus, parietal cortex, occipital cortex and frontal cortex, and lowest in white matter. Western blotting results showed that the content of BDNF protein in the cerebrum of yaks was the highest in temporal cortex,followed by hippocampus. The content of BDNF protein in other tissues was parietal cortex, frontal cortex and cerebrum white matter, and the content of BDNF protein was the lowest in occipital cortex. The content of BDNF protein intlecerebrum of cattles was the highest in the temporal cortex, followed by the hippocampus. The content of BDNF protein in other tissues was parietal cortex, occipital cortex and frontal cortex in descending order, and the protein content in cerebrum white matter was the lowest. Immunohistochemical results showed that the positive expression of BDNF protein in the cerebrum of yaks and cattles was basically similar, mainly distributed in the granulosa cells and glial cells in the frontal cortex, temporal cortex, parietal cortex and occipital cortex, glial cells in cerebrum white matter, pyramidal cell layer and polyform cell layer in the hippocampus. There was the small amount of distribution in Martinotti cells and the molecular layer of hippocampus in the cerebral cortex. Conclusion BDNF mRNA and protein are distributed and expressed in different brain regions of yaks and cattles, but the expression level different, which is speculated to be closely related to the specific functions of different cerebrum regions. The expression level of the cerebrum of yak is higher than that of cattle except occipital cortex, suggesting that it is related to the altitude hypoxic environment. BDNF may play an important role in enhancing hypoxic tolerance and protecting internal environmental homeostasis in the process of animal adaptation to hypoxic environment.
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Objectives@#In the Philippines, patients on chronic hemodialysis with COVID-19 remain admitted in hospitals despite clinical recovery because most free-standing dialysis units require proof of negative conversion via Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). This study aims to determine the time to negative conversion of COVID-19 RT-PCR testing among adult patients on chronic hemodialysis with COVID-19 admitted at the Philippine General Hospital (PGH) and bring insight in using the symptom or time-based procedure as recommended by local guideline, and ultimately, to ensure delivery of adequate hemodialysis despite being infected with COVID-19, shorten isolation period, and conserve resources especially in resource-limited settings.@*Methods@#This is a retrospective cohort study on all adult patients on chronic hemodialysis who were admitted in PGH after the diagnosis of COVID-19 by RT-PCR between March 2020 and February 2021. Descriptive statistics was used in summarizing the data.@*Results@#A total of 90 patients on chronic hemodialysis who tested positive for COVID-19 via RT-PCR admitted at PGH were included in the study. Most of these patients had moderate COVID-19 at 53.3%. The median number of days from onset of symptoms to clinical recovery was 14.5 days. The median time to first negative conversion was 18 days. Most of these patients had negative conversion at the second week. The correlation coefficient between time to clinical recovery and negative conversion was 0.214. @*Conclusion@#Among adult patients on chronic hemodialysis who were admitted in PGH after the diagnosis of COVID-19, the time to negative conversion was longer compared to the time to clinical recovery with a very weak correlation between the two.
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COVID-19 , Renal DialysisABSTRACT
@#Objective To develop and verify a multiplex fluorescent quantitative PCR method for detection of common bacterial and fungal contaminants in cell culture medium.Methods According to NUC gene of Staphylococcus aureus,COLA gene of Clostridium spore,ITS-2 segment sequence of Candida albicans,a set of primers and probes were designed for each respectively,and using UBI3 gene of capsicum introduced as external standard gene,a triple reaction system of Staphylococcus aureus,Clostridium spore and external standard gene and a double reaction system of Candida albicans and external standard gene were established.The primer specificity,linear range,limit of detection,specificity,anti-interference performance and precision of the method were verified.Finally,100 samples of 293T cell culture medium were detected by using the developed method,which was compared with the common PCR method.Results Three pairs of primers all amplified about 100 bp specific gene bands corresponding to the three strains at different annealing temperatures(56,57,58 and59 ℃),and the size was consistent with the expected.In the range of 5.80 × 10~6 — 5.80 × 10~2 copies/μL,the standard plasmids of the three strains showed a good linear relationship with the Ct values.The standard curve equations were:Y=-3.373 X+37.48,Y=-3.557X+36.59 and Y=-3.536 X+39.78,each R~2> 0.99,respectively,and the amplification efficiency was in the range of 90%—110%.All the limits of detection of the three strains were 10~1 CFU/mL.The primers and probes of the three strains showed no specific amplification on the genomic DNA of six kinds of cells that were prone to cross-reaction.The genomic DNA of 293T cells,Yeast,Escherichia coli and Mycoplasma sp.had no effect on the detection.The CVs of repeatability and intermediate precision verification were both less than 15%.Among 100 cell culture medium samples,14 positive and 86 negative samples were detected,and the results of common PCR method for three positive and two negative samples randomly selected were consistent with the developed method.Conclusion The multiplex fluorescent quantitative PCR method developed in this study for the detection of bacteria and fungi in cell culture medium has good specificity,anti-interference performance and precision,and is simple to operate with low cost and high sensitivity,which can quickly detect the contaminants during cell culture.
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@#Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to approximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry, and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280) ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD) of 4.2%.The 95% confidence interval for the mean was 103.8—105.8 ng/μL,and the 95% reference range for single measurements was 96.0—113.6 ng/μL.The average confidence limit rate was 1.0%,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3—3 000 pg/reaction,with amplification efficiencies of 101% and 95%,and R~2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.
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Organ transplantation has become an effective treatment for multiple end-stage diseases. However, the recipients of organ transplantation need to take immunosuppressive drugs for a long time after operation, which leads to low immune function and relatively high incidence of bacterial, viral and fungal infections. Traditional microbial detection methods, such as pathogen culture, immunological detection and polymerase chain reaction, have been widely applied in infection detection, whereas these methods may cause problems, such as long detection time and presumed pathogens. Metagenomic next-generation sequencing has been widely adopted in infection prevention and control in organ transplantation in recent years due to high detection rate and comprehensive detection of pathogen spectrum. In this article, the application of metagenomic next-generation sequencing in the prevention and control of infection in solid organ transplantation was reviewed, aiming to provide reference for the diagnosis and treatment of transplantation-related infection.
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'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.
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Animals , Anthocyanins , DNA Shuffling , Flowers/genetics , Porifera , Prunus/genetics , Glutamine/analogs & derivatives , Plant ExtractsABSTRACT
Neoadjuvant therapy is a preoperative systemic treatment for patients with breast cancer. This therapy has greatly improved the clinical outcomes of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, which is associated with poor prognosis. Currently, dual anti-HER2 antibodies, including trastuzumab and pertuzumab, combined with non-anthracycline chemotherapy is one of the standard regimens to achieve high pathologic complete response rate and satisfactory efficacy. The combination of trastuzumab with tyrosine kinase inhibitors, antibody-drug conjugate drugs, or immunotherapy combined with target therapy, under the indications of reasonable biomarkers, is effective for HER2-positive breast cancer. In this article, we briefly reviewed neoadjuvant therapy in the dual-targeting therapy era and discussed its future perspectives.
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@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines
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ObjectiveTo establish an allele-specific polymerase chain reaction （PCR） method for identifying Scolopendra dispensing granules， so as to ensure the quality and therapeutic effects of Scolopendra and its preparations. MethodThe primer interval suitable for the PCR was selected based on the cytochrome c oxidase subunit 3（COX-3） gene sequence of Scolopendra， and the single nucleotide polymorphism （SNP） loci of Scolopendra and its adulterants were mined from the interval for the design of specific primers. The samples of Scolopendra and its adulterants were collected. The PCR system was established and optimized regarding the annealing temperature， cycles， Taq enzymes， DNA template amount， PCR instruments， and primer concentrations， and the specificity and applicability of this method were evaluated. ResultThe PCR system was composed of 12.5 μL 2×M5 PCR Mix， 0.4 μL forward primer （10 μmol·L-1）， 0.4 μL reverse primer （10 μmol·L-1）， 2.5 μL DNA template， and 9.2 μL sterile double distilled water. PCR parameters： Pre-denaturation at 94 ℃ for 3 min， 30 cycles （94 ℃ for 20 s， 62 ℃ for 20 s， 72 ℃ for 45 s）， and extension at 72 ℃ for 5 min. After PCR amplification with the system and parameters above， the electrophoresis revealed a bright band at about 135 bp for Scolopendra and no band for the adulterants. ConclusionThe established allele-specific PCR method can accurately identify the medicinal materials， decoction pieces， and standard decoction freeze-dried powder of Scolopendra， as well as the intermediates and final products of Scolopendra dispensing granules， which is of great significance for ensuring the quality and clinical efficacy of Scolopendra and its preparations.
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ObjectiveTo establish a polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method for rapid distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex， so as to avoid the influence of genetic confusion on drug safety. MethodThe DSS-tagged sequences of Periplocae Cortex were obtained from the Chloroplast Genome Information Resource （CGIR） and analyzed to find the enzymatic cleavage sites that were different from those of Acanthopanacis Cortex and Lycii Cortex. The specific enzymatic cleavage site， Cla I， of Periplocae Cortex was selected， on the basis of which the primers for PCR-RFLP were designed. Furthermore， the factors such as annealing temperature， number of cycles， Taq enzyme， PCR instruments， and enzymatic treatment time that may influence PCR-RFLP were studied. The established PCR-RFLP method was applied to the identification of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex samples produced in different regions. ResultThe PCR-RFLP at the annealing temperature of 59 ℃ and with 40 cycles showed clear bands of the samples. When the enzyme digestion time was 30 min. The reaction produced the target bands at about 140 bp and 290 bp for both Periplocae Cortex and its original plant and only a band at about 430 bp for Acanthopanacis Cortex， Lycii Cortex， and their original plants. The method can accurately distinguish Periplocae Cortex from its confounders Acanthopanacis Cortex and Lycii Cortex. ConclusionThe PCR-RFLP method for distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex was established. It has high stability， sensitivity， and applicability， providing a reference for the quality control of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex.
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ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.
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ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.
ABSTRACT
Abstract Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.
Resumo Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.