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【Objective】 To investigate the effectiveness of multilink real-time fluorescence quantitative PCR (qPCR) in the detection of common pathogens in transplantation. 【Methods】 The primers of the qPCR detection system were designed for 24 common infectious pathogens after clinical transplantation, and the standard plasmids of each pathogen were used to verify the qPCR reaction.After the primer probe effect and concentration of each pathogen reaction system in this experiment was optimized, the sensitivity, correlation coefficient (R2) and amplification efficiency (E) of qPCR method were analyzed and confirmed.Twenty-two samples from patients, who underwent liver and kidney transplantation in transplant ICU of Sichuan Provincial People′s Hospital, were used to verify the application of the detection system.The total nucleic acid of 100 μL was extracted from each individual and divided into two aliquots, which were detected by multi-link qPCR reaction system and analyzed by high-throughput sequencing method (NGS). At the same time, samples (2 mL each) were taken from the transplanted patients for microbial culture.The results of the three detection methods were compared, and the NGS method was taken as the gold standard to analyze the positive detection rate of the multi-link qPCR method and its difference with the culture method and NGS. 【Results】 The lower limit of qPCR detection for 24 pathogens in the established qPCR detection system was 101cp/μL(R2>0.99), with the positive rate of pathogens at 59.1% (13/22), showing significant difference versus microbial culture (18.2%, 4/22)(P<0.05), but not versus NGS (63.6%, 14/22)(P>0.05). Percentage of pathogens detected was as follows: human herpetic virus type 6 (HHV-6) 30.8% (4/13), cytomegalovirus (HCMV) 23.1% (3/13), Epstein-Barr virus (EBV) 23.1% (3/13), human parvovirus B19 15.4% (2/13), Haemophilus influenzae (Hin) 15.4% (2/13), Enterococcus faecium (EFM) 15.4% (2/13), Clostridium difficile 15.4% (2/13), Escherichia coli 7.7% (1/13), Stenotrophomonas maltophilia (Sma) 7.7% (1/13), Klebsiella pneumoniae (Kpn) 7.7% (1/13), Enterococcus faecalis (Efa) 7.7% (1/13) and Streptococcus pneumoniae (Spn) 7.7% (1/13). The consistency rate of pathogens detected by the three methods was 32% (7/22), among which the consistency rate of multi-link qPCR with NGS method was 59% (13/22), and multi-link qPCR with microbial culture was 41% (9/22). 【Conclusion】 Compared with the microbial culture, the multi-link qPCR method demonstrated high sensitivity, accurate quantification, short time and low cost for the detection of common pathogens in clinical transplantation.Multi-link qPCR combined with NGS and microbial culture is helpful to quickly predict the pathogen infection status of patients after transplantation.
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@#There are many ways to extract and culture neural stem cells in vitro, but the viability and stability of neural stem cells obtained by different methods are different. By thinking about the process of extracting and culturing neural stem cells in vitro from the cerebral cortex of SD fetal rats, we summarized extraction steps, the main points of extraction, the selection basis of culture medium, selection of inoculation density, cultural method, methods of solution changing, passage time and passage methods. At last a large number of neural stem cells with high vitality and stability have been obtained and applied to the basic research of neural stem cells.
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Objective: To explore the solid fermentation process of Panax ginseng by Monascus purpureus, which can transfer some major ginsenoside into rare ginsenoside Rg3 with stronger biological activity. Methods: The static dark culture method was used to perform microbial fermentation; Vanilline-glacial acetic acid method was used to detect the total saponins before and after fermentation, and the ginsenoside Rg3 was detected by HPLC. Results: The optimum process parameters of Monascus purpureus fermentation was fermentation 6 d, fermentation temperature 32 ℃, pH 7.0, and water content of substrate 50%. After 6 d of fermentation, the content of total saponins in fermentation products increased by 40%, and the content of ginsenoside Rg3 was 6.047 mg/g, which was 2.3 times as much as that of non-fermented P. ginseng. According to the change of monomer saponin content along with the fermentation time, it was deduced that the transformation path was Rb1 (Rb2)→Rd→Rh2→Rg3. Conclusion: The solid fermentation process of Monascus purpureus established in this study is reasonable, which not only lays a foundation for the directional production of rare saponins Rg3 but provides a theoretical support for preparing rare ginsenoside in vitro.
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Objective@#To investigate the value of real-time RNA simultaneous amplification and testing (SAT) in the detection of Ureaplasma urealyticum (UU) in the semen of infertile males and its clinical significance.@*METHODS@#We collected semen samples from 542 infertility patients and 120 normal fertile men as controls in the Andrology Clinic of Nanjing General Hospital from March to September 2015. We detected UU infection in the samples using the culture method and SAT technology, respectively.@*RESULTS@#All the UU positive cases (except 4 false positive cases) detected by the culture method were also shown to be positive in SAT. The UU detection rate of SAT was significantly higher than that of the culture method both in the infertility patients (54.1 vs 19.7%, P<0.05) and in the normal controls (42.5 vs 12.5%, P<0.05).@*CONCLUSIONS@#SAT is a rapid and accurate method for detecting UU infection in semen samples, with a higher sensitivity and accuracy than the culture method, and it can also be used to evaluate the therapeutic effects. However, the culture method has its own advantages, such as low requirement of technical equipment, easy operation, and possibility of drug sensitivity test at the same time. Therefore, SAT and the culture method can be used alternatively according to the clinical need.
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Humans , Male , Andrology , Infertility, Male , Microbiology , Nucleic Acid Amplification Techniques , RNA, Bacterial , Semen , Chemistry , Microbiology , Semen Analysis , Ureaplasma Infections , Diagnosis , Ureaplasma urealyticum , GeneticsABSTRACT
Objective To analyze the characteristics of toxin, the PCR-ribotyping(RT) and the multilocus sequence typing(MLST) of Clostridium difficile strains isolated from China-Japan Friendship Hospital in order to provide a basis for monitoring the outbreak of nosocomial Clostridium difficile infection.Methods A total of 321 samples were collected from the patients with suspected Clostridium difficile infection(CDI) in China-Japan Friendship Hospital(CJFH) during 2012 to 2013.All Clostridium difficile strains were isolated and identified by the standard phenotypic culture method.Cytotoxicity test was performed to detect toxin B.Toxin genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) harbored by those strains were analyzed.RT and MLST were used for homologous analysis.Clinical data of the patients were collected to analyze the isolation rate of Clostridium difficile in different populations.Results Forty-eight strains of Clostridium difficile were isolated from 46 patients with diarrhea and three of them were isolated from the same patient.The incidence of CDI among all patients, outpatients and inpatients were 14.3%(46/321), 12.8%(5/39) and 14.5%(41/282), respectively.Toxin B was detected in all of the strains as indicated by the cytotoxicity test.Strains of sequence type 1(ST1) showed the strongest cytotoxicity of all the isolated Clostridium difficile strains.Ten out of the 48 strains (20.8%) were tcdA(-)/tcdB(+) strains, which belonged to either ST37 or ST81.The results of RT and MLST were consistent in assigning the strains into nine types, in which the predominant type was ST1/RT027 accounting for 27.1% (13/48).All of the ST1/RT027 strains presented a toxin gene profile of tcdA(+)/tcdB(+) and cdtA(+)/cdtB(+).Most of the ST1/RT027 strains were isolated from the Traditional Chinese Medicine Department of Respiratory, where smallnosocomial outbreaks of ST1/RT027 strain infection might happen.Conclusion CDI diagnosed in CJFH mainly belongs to nosocomial infection.Most of the isolated strains harbor tcdA(+)/tcdB(+) genes.Surveillance for the outbreaks of CDI caused by ST1/RT027 strains over producing toxins A and B should be strengthened in hospitals.
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AIM:To explore a novel method to isolate human nucleus pulposus mesenchymal stem cells (hNP-MSCs) in vitro and to identify their biological characteristics.METHODS:The explant culture method was employed to isolate hNP-MSCs from nucleus pulposus tissue obtained by percutaneous endoscopic lumbar discectomy (PELD).The isolated cells were passaged for purification and cultured in vitro followed by morphological observation.The cell proliferation ability was detected by CCK-8 assay.Growth curves of the cells were drawn and surface antigens were detected by flow cytometry.The cells at the 3rd~6th passages were induced for adipogenic, osteogenic and chondrogenic differentiation, and examined by oil red O staining, alizarin red staining and Alcian blue staining.RESULTS:The cells with self-renewal were obtained from nucleus pulposus tissue obtained by PELD.The results of flow cytometry analysis revealed that the cells were positive for CD29, CD44, CD90, CD73 and CD105, but negative for CD34 and CD45.The proliferative capacity was consistent with the growth characteristics of MSCs and multilineage differentiation potential was identified.CONCLUSION:A novel method to efficiently isolate and culture hNP-MSCs,PELD combined with explant culture method,was established, which would promote the study of regenerative medicine based on hNP-MSCs.
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Objective:To isolate SD rat adipose-derived stem cells(ASCs)by suspended explant culture method.Methods: The healthy rat inguinal fat pads were obtained.The SD rat ASCs were isolated by suspended explant culture method and explant adherent culture method.The growth status and morphology were observed.The growth curve and cell surface markers CD29,CD44,CD45 of the 3rd passage cells were analyzed respectively by CCK-8,immunocytochemistry;the 3rd passage cells were induced individually by adipogenic differentiation medium and osteogenic differentiation medium,and cells were examined by oil red O staining and alizarin red staining.Results: The SD rat ASCs obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curves were typical of S type.Immunocytochemistry analysis revealed that the third passage of SD rat ASCs were positive for CD29,CD44,but were negative for CD45.SD rat ASCs were positive for oil red O staining at 14 days after adipogenic induction,and positive for alizarin red staining at 14 days after osteogenic induction.Conclusion: Isolation of SD rat ASCs by suspended explant culture method is successfully established.The method is simple.It provides a new method for the isolation of SD rat ASCs in vitro.
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Objective To compare the capabilities of culture method, polymerase chain reaction ( PCR) and serological test in identifying Mycoplasma pneumoniae infection in children with confirmed com-munity acquired pneumonia. Methods Bronchoalveolar lavage fluid and serum samples were collected from hospitalized children with community acquired pneumonia in Capital Institute of Pediatrics from March to May in 2016. Three methods, traditional culture method, PCR and serological test, were respectively used to de-tect Mycoplasma pneumoniae infection in those children. Statistical analysis was performed by using SPSS18. 0 software and chi-square test. Results Seventy-nine children with community acquired pneumonia were enrolled in this study. Eight (10. 13%) patients were diagnosed with Mycoplasma pneumoniae infec-tions by the traditional culture method with an average positive culture period of 21 days. Twenty-three (29. 11%) patients showed positive results by using PCR analysis, including the 8 patients identified by the culture method. Forty-one (51. 90%) patients were found to be positive for Mycoplasma pneumoniae infec-tions by the serological test. However, four negative samples identified by the serological test were confirmed to be positive by PCR analysis, including two positive samples confirmed by the culture method. Statistical analysis showed that the differences in positive rates detected by using the three methods were statistically significant. Conclusion It is recommended that both serological test and PCR analysis should be used in combination with clinical symptoms for a comprehensive assessment of Mycoplasma pneumonia infection in children.
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Legionella spp. es una bacteria ambiental capaz de sobrevivir en un amplio intervalo de condiciones fisicoquímicas y puede colonizar los sistemas de distribución y almacenamiento del agua potable. Legionella pneumophila es el principal patógeno trasmitido por el agua y produce el 90% de los casos de legionelosis. El objetivo del presente trabajo fue detectar por cultivo la presencia de Legionella spp. en depósitos domiciliarios de agua potable de la ciudad de Resistencia, Chaco. La detección de Legionella en las muestras de agua se realizó por cultivo según lo establecido en la norma ISO 11731:1998. Se analizaron 32 muestras de agua y de 12 (37,5%) de ellas se recuperaron cepas de Legionella spp. La vigilancia de este microorganismo en el agua de consumo humano representa el primer paso para controlar su diseminación hacia huéspedes susceptibles.
Legionella spp. is an environmental bacterium that can survive in a wide range of physicochemical conditions and may colonize distribution systems of drinking water and storage tanks. Legionella pneumophila is the major waterborne pathogen that can cause 90% of Legionnaires' disease cases. The aim of this study was to detect the presence of Legionella spp. in household drinking water tanks in the city of Resistencia, Chaco. The detection of Legionella in water samples was performed by culture methods as set out in ISO 11731:1998. Thirty two water samples were analyzed and Legionella spp. was recovered in 12 (37.5%) of them. The monitoring of this microorganism in drinking water is the first step towards addressing the control of its spread to susceptible hosts.
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Drinking Water/analysis , Legionellosis/prevention & control , Legionella pneumophila/isolation & purification , Legionella pneumophila/pathogenicity , Water Pollution/analysis , Drinking Water/microbiology , Surveillance in DisastersABSTRACT
Objective:To isolate rabbit fibroblast-like synoviocytes(FLSs) by suspended explant culture method. Methods:The healthy rabbit joint synovial layers were obtained. The cells were cultured by suspended explant culture method and compared with the explants adherent culture method. The growth status and morphology were observed. The growth curve of the 3rd passage cells was measured by CCK-8 assay,and the expression of vimentin was tested by immunocytochemistry. Results: The FLS obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand. The cell growth curve was typical of S type, and the cells highly expressed vimentin. Conclusion:Primary culture of rabbit fibroblast-like synoviocytes by suspended explant culture method were successfully established. The method was simple and highly efficient. It provided a new method for the isolation of FLS in vitro.
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Objective To establish an easier and better method for primary culture of rabbit fibroblast-like synoviocytes(FLS) by modified tissue cell culture.Method Healthy rabbit joint synovial layers were cut into small pieces and siphoned off water attached on the tissue with sterile filter paper and then cultured.The growth status and morphology were observed.The growth curve of the 3rd passage cells was measured by CCK-8 assay, and the expression of vimentin was detected by immunocytochemistry.Results The FLS cultured in vitro exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curve was typical of S type, and the cells highly expressed vimentin.Conclusions Primary culture of rabbit FLS by the improved explant culture method is successfully established.The method is simple and highly efficient.It provideds a new method for isolation of FLS.
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Adipose-derived stem cells ( ASCs ) as potential seeded cells have been widely used in tissue engineering.Thus to obtain enough, high activity, high purity adipose-derived stem cells is the particular important premise of the application in tissue engineering.In this paper, the isolation and purification methods of ASCs were reviewed and the merit and demerit of different methods were compared in order to provide theoretical basis for safe and high-effective isolation and purification of ASCs.
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BACKGROUND/AIMS: Peritonitis is the most frequent complication of continuous ambulatory peritoneal dialysis (CAPD). Prompt recognition and treatment of peritonitis is important. The purpose of this study was to compare the effectiveness of isolation of the microorganisms causing CAPD peritonitis by the BACTEC blood culture and conventional methods. METHODS: We retrospectively reviewed 38 episodes of peritonitis in 34 CAPD patients between September 2007 and February 2010. Two methods of processing dialysate from patients on CAPD were used. Blood culture was performed using two 10-mL effluents, which were inoculated into a pair of BACTEC culture bottles. The conventional method was performed using 50 mL of centrifuged dialysate. The sedimented dialysate was inoculated onto blood agar and MacConkey agar plates or into thioglycollate broth. To evaluate effectiveness, we compared the rate of positive culture results and the time to identify the causative organism of the two culture methods. RESULTS: Use of the BACTEC bottle method resulted in more positive culture results than did conventional culture (86.8 vs. 57.9% p = 0.003). The time taken to identify the causative organism from culture-positive peritonitis was more rapid using the blood culture compared with the conventional culture method (90 vs. 109 hr, p = 0.03). CONCLUSIONS: Blood culture using the BACTEC bottle is more effective than the conventional culture technique for detection of causative microorganisms in CAPD peritonitis.
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Humans , Agar , Culture Techniques , Peritoneal Dialysis, Continuous Ambulatory , Peritonitis , Retrospective StudiesABSTRACT
Objective:To establish a better method for isolating and culturing preadipocytes from rat white adipose tissue.Methods:The adipose tissue was digested by collagenⅠand preadipocytes were collected by centrifugations and filtrations.Cells were cultured using basic and differentiation media.Preadipocytes were detected by morphologic method,auxodrome study and stained with Oil red O for determining the cell identity.Results:Using our modified method,primary preadipocytes can be harvested in greater amount compared with the original method.The primary preadipocytes could differentiate into adipocytes under the induction of differentiation medium.The preadipocytes obtained by our method had the characteristics of preadipocytes.Conclusion:We have established an efficient preadipocyte culture method.Our medication method is better than the original method.
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AIM: To explore a long-term Kupffer cell culture method, which can best maintain the function and activity of cell and decrease the cellular damage. METHODS: Lactic dehydrogenase (LDH) in the supernatants were measured to observe the severity of cellular damage, the function of Kupffer cell was reflected with the nitrogen monoxide(NO) content and the intracellular acid phosphatases(ACP) activity. In the conditions of common culture(CC), single collagen culture(SCC) and double collagen culture(DCC), with or without serum, study the maintenance of cellular function and the severity of cellular damage. RESULTS: Compared with the other two methods, the LDH contents were significantly lower (P < 0.05; P < 0.01), the levels of both NO at different days and the cellular ACP at 15d were markedly increased (P < 0.05; P < (0.01) in DCC group with serum. Even without serum, the cellular damage and function could achieve satisfactory results in DCC group. CONCLUSION: DCC is a useful method to study Kupffer cell in vitro.
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BACKGROUND: Peripheral blood is the most frequently used specimen for routine chromosome analysis. The classical method using flask for cell culture needs a lot of reagent and is cumbersome. To simplify the procedure, we tried using culture tube and modifying the fixation method. The purpose of this study is to assess the reliability of the modified method using culture tube for chromosome analysis of peripheral blood. METHODS: We tested peripheral blood of ten normal healthy persons. In the modified method, culture tube (110 mm 16 mm, Nunc, Roskilde, Denmark) containing 2.25 mL RPMI 1640 supplemented with fetal calf serum and phytohemagglutinin was used. Harvest was done in the culture tube. Fixative 2.0 mL was added without centrifugation after hypotonic treatment. After 10 min incubation at room temperature, the cells were pelleted and washed. This method was compared with the reference method using flask. We evaluated the quality of chromosome and calculated mitotic index. RESULTS: Chromosome quality of the modified method using culture tube was good and the same as that of the reference method. Mitotic index was higher in the modified method (1.0~4.3%, mean 2.5%) than in the reference method (0.4~2.2%, mean 1.2%) (P<0.01). CONCLUSIONS: The modified method needs less amount of reagent and is easy to do. The chromosome quality was good enough to evaluate the karyotype. So, this modification enable to improve the effectiveness of chromosome laboratory.
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Humans , Cell Culture Techniques , Centrifugation , Karyotype , Mitotic IndexABSTRACT
BACKGROUND: The techniques that are currently used to diagnose nail infections, KOH and culture, can only provide indirect evidence of a fungal cause because false-negative and falsepositive results are high. The use of histologic examination can be of help for a more accurate and specific diagnosis. OBJECTIVE: This study was undertaken to investigate the characteristics of nail invasion and morphology in nail sections by 5 species of fungi including Trichophyton mentagrophutes var mentagrophytes, Trichophyton, rubrum, Candida albicans, Scopulariopsis brevicaulis, and Fusarium oxysporum. METHODS: Two in vitro methods for the study of nail invasion were used. In one method, those cultured fungi were inoculated on the ventral surface of the human nail clippings (direct nail inoculation method). In the other method, invasion of nail clippings by those fungi was induced in the continuous shaking liquid media (continuous shaking liquid culture method). RESULTS: 1. In direct nail inoculation method, the gross findings are similar to those obtained in routine culture media. By 1 week, the nail fragments were totally covered by a white fungal mycelium on gross examination. 2. Non-dermatophytes were slower invader of nail tissue than dermatohytes. Invasion was quicker and more extensive in the dystrophic nail. Full thickness invasion of the normal nail fragment was observed in 46.8+/-9.8 days. But it took 13.3+/-2.6 days to invade the dystrophic nail fragment (p<0.05). 3. This model showed the morphologic differences of three groups of fungi. Deramtolphytes gernerally showed regular, straight, septate and branched hyphae, which run parallel to the nail surface; C. albicans appeared as pseudofilaments running haphazardly within the nail; S. brevicaulis and F. oxysporum appeared as irregular, thicker hyphae without any spores. 4. By using the continuous shaking liqyid culture method, T. Mentagrophytes var mentagrophytes was only successful in nail invasion. CONCLUSION: The direct nail inoculation method is a simple method showing the dynamics of the nail invasion in vitro. Unlike to dermatolphytes, NDFF(non-dermatolphytic filamentous fungi) and Candida sp. could invade only dystrophic abnormal nail. Dermatophytes, Candida sp., and NDFF showed some differences in shape and arrangement fo the hyphae on the histopathologic sections. But they are not diagnostic to the species.
Subject(s)
Humans , Arthrodermataceae , Candida , Candida albicans , Culture Media , Diagnosis , Fungi , Fusarium , Hyphae , Mycelium , Nails, Malformed , Running , Scopulariopsis , Spores , TrichophytonABSTRACT
According to the yield of intracellular triterpene, the strain GL31 was screened out from 22 strains of Ganoderma collected from various regions. The optimal fermentation conditions were studied by single factor experiments and orthogonal test. The optimal parameters of carbon source, nitrogen source, initial pH, the medium volume in the flask and cell density were obtained. In addition, the metabolic curves of intracellular triterpene and biomass were determined. It was found that the yield of intracellular triterpenes was up to 3.51?10~(-2)g/100 mL medium at the 84th hour. In addition the stram was staticly fermented 144 hours flowing 84hours shaking-flask culture, the IT (intracellular triterpenes) content was increased by 48.6% and the yield of IT was increased by 65% compared with those of the mycelia cultured 84hours using shaking-flask method. This indicated that static fermentation method is helpful for improving the total IT content. The result also show that the highest yield of intracellular triterpenes of mycelia staticly fermented flowing 84hours shaking-flask culture is higher than that of mycelia using shaking flask culture method.
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AIM To explore a long-term Kupffer cell culture method, which can best maintain the function and activity of cell and decrease the cellular damage. METHODS Lactic dehydrogenase (LDH) in the supernatants were measured to observe the severity of cellular damage ,the function of Kupffer cell was reflected with the nitrogen monoxide(NO) content and the intracellular acid phosphatases(ACP) activity. In the conditions of common culture(CC),single collagen culture(SCC) and double collagen culture(DCC), with or without serum, study the maintenance of cellular function and the severity of cellular damage. RESULTS Compared with the other two methods, the LDH contents were significantly lower( P