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Objective@#To explore the preliminary application effect of real-time fluorescence recombinase polymerase amplification (RPA) in the detection of Candida albicans.@*Methods@#(1) Candida albicans standard strain and negative control bacteria of Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Candida glabrata standard strains of respectively 1 mL were collected and their DNA were extracted by yeast/bacterial genomic kit. The specificity of polymerase chain reaction (PCR), real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. (2) One Candida albicans standard strain and one negative control bacteria of Candida glabrata standard strain were collected, resuscitated, and counted. Candida albicans was diluted 10 times to 1×107 to 1×101 colony-forming unit (CFU)/mL. The DNA of the two bacteria were extracted as experiment (1). The sensitivity of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting Candida albicans were analyzed. The number of cycles for amplification curve to reach the threshold in real-time fluorescent quantitative PCR, and time of appearance of specific amplification curve in real-time fluorescence RPA were recorded and compared with the results in PCR. The detection limit and rate of the above-mentioned 3 methods in detecting Candida albicans were analyzed, and the correlation between concentration of Candida albicans in real-time fluorescence RPA and detection time was analyzed. (3) One standard strain of Candida albicans was collected, and the DNA was extracted as experiment (1) and detected by PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA. The total detection time of the above-mentioned 3 methods was recorded, respectively. (4) The DNA of 31 clinical samples of suspected Candida albicans infection and 1 clinical sample of Candida albicans collected from cotton swab were extracted, PCR and real-time fluorescence RPA were carried out, and the positive detection rates of the above-mentioned methods were calculated. The DNA of the clinical samples with positive results in both PCR and real-time fluorescence RPA were extracted by yeast/bacterial genomic kit, chelex-100 boiling method, and repeatedly freeze-thawing with liquid nitrogen method, and real-time fluorescence RPA and PCR were carried out. The negative control bacteria was Candida glabrata in real-time fluorescence RPA, while negative control bacteria in PCR were the same as experiment (1). The positive results in PCR and real-time fluorescence RPA were observed and time for amplification curve to reach the fluorescence threshold in real-time fluorescence RPA was recorded, respectively. Data were processed with linear correlation analysis and t test.@*Results@#(1) Three methods showed positive results in detecting standard strain of Candida albicans, and none of the 5 negative control bacteria showed positive results. (2) As the concentration of bacterial solution of Candida albicans decreased, the number of cycles for the amplification curve to reach the threshold increased in real-time fluorescent quantitative PCR, the time for appearance of specific amplification curve prolonged in real-time fluorescence RPA, and brightness of the gel strip weakened in PCR. None of the negative control bacteria in the above-mentioned 3 detection methods showed corresponding positive results. The detection limit of Candida albicans in real-time fluorescence RPA, PCR, and real-time fluorescent quantitative PCR was 1×101 CFU/mL. There was a significant negative correlation between the concentration of Candida albicans and the detection time in real-time fluorescence RPA (r=-0.95, P<0.01). The positive detection rates of PCR and real-time fluorescent quantitative PCR for Candida albicans of 1×101 to 1×107 CFU/mL were 100%. The positive detection rate of real-time fluorescence RPA for Candida albicans of 1×101 CFU/mL was 78%, and the positive detection rate of real-time fluorescence RPA for Candida albicans of 1×102 to 1×107 CFU/mL was 100%. (3) The total time of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA detection for Candida albicans was 133, 93, and 35 min, respectively. (4) The positive detection rate of real-time fluorescence RPA for 31 clinical samples of suspected Candida albicans infection was 32.26% (10/31), which was slightly lower than 35.48% (11/31) of PCR. Eleven clinical samples showed positive results both in real-time fluorescence RPA and PCR detection. No positive result was observed in the negative control bacteria detected both by real-time fluorescence RPA and PCR. The DNA was extracted by yeast/bacterial genomic extraction kit and chelex-100 boiling method for real-time fluorescence RPA detection. The time for the amplification curve to reach the threshold was (438±13) and (462±12) s, respectively, which was close (t=1.32, P>0.05). The DNA was extracted by repeatedly freeze-thawing with liquid nitrogen method for real-time fluorescence RPA, and the time for the amplification curve to reach the threshold in real-time fluorescence RPA was (584±15) s, which was significantly longer than that in the other 2 methods (t=7.55, 6.39, P<0.01).@*Conclusions@#Real-time fluorescence RPA has advantages of rapid detection, simple operation, high sensitivity, and good specificity in detecting Candida albicans, which is worthy of clinical application.
ABSTRACT
Objective@#To establish an optimized method of recombinase polymerase amplification (RPA) to rapidly detect Pseudomonas aeruginosa in clinic.@*Methods@#(1) The DNA templates of one standard Pseudomonas aeruginosa strain was extracted and detected by polymerase chain reaction (PCR), real-time fluorescence quantitative PCR and RPA. Time of sample loading, time of amplification, and time of detection of the three methods were recorded. (2) One standard Pseudomonas aeruginosa strain was diluted in 7 concentrations of 1×107, 1×106, 1×105, 1×104, 1×103, 1×102, and 1×101 colony forming unit (CFU)/mL after recovery and cultivation. The DNA templates of Pseudomonas aeruginosa and negative control strain Pseudomonas putida were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (3) The DNA templates of one standard Pseudomonas aeruginosa strain and four negative control strains (Staphylococcus aureus, Acinetobacter baumanii, Candida albicans, and Pseudomonas putida) were extracted separately, and then they were detected by PCR, real-time fluorescence quantitative PCR, and RPA. The specificity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (4) The DNA templates of 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain Pseudomonas putida were extracted separately, and then they were detected by RPA. Positive amplification signals of the clinical strains were observed, and the detection rate was calculated. All experiments were repeated for 3 times. Sensitivity results were analyzed by GraphPad Prism 5.01 statistical software.@*Results@#(1) The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting Pseudomonas aeruginosa were all 20 minutes. In PCR, time of amplification was 98 minutes, time of gel detection was 20 minutes, and the total time was 138 minutes. In real-time fluorescence quantitative PCR, amplification and detection could be completed simultaneously, which took 90 minutes, and the total time was 110 minutes. In RPA, amplification and detection could also be completed simultaneously, which took 15 minutes, and the total time was 35 minutes. (2) Pseudomonas putida did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of Pseudomonas aeruginosa in real-time fluorescence quantitative PCR and PCR was 1×101 CFU/mL, and that of Pseudomonas aeruginosa in RPA was 1×102 CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of Pseudomonas aeruginosa, the shorter threshold time and smaller the number of cycles, namely shorter time for detecting the positive amplified signal. In real-time fluorescence quantitative PCR, all positive amplification signal could be detected when the concentration of Pseudomonas aeruginosa was 1×101-1×107 CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of Pseudomonas aeruginosa was 1×101 CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of Pseudomonas aeruginosa was 1×102 CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of Pseudomonas aeruginosa was 1×103-1×107 CFU/mL. (3) In RPA, PCR, and real-time fluorescence quantitative PCR, Pseudomonas aeruginosa showed positive amplification signals and gel positive results, but there were no positive amplification signals or gel positive results in four negative control strains of Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas putida. (4) In RPA, 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin and 1 clinical strain of Pseudomonas aeruginosa taken by cotton swab showed positive amplification signals, while Pseudomonas putida did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of Pseudomonas aeruginosa in RPA was 100%.@*Conclusions@#The established optimized RPA technology for fast detection of Pseudomonas aeruginosa requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of Pseudomonas aeruginosa infection in clinic.
ABSTRACT
Different harvest times of Isatidis Folium had a significant effect on the yield and the quality of Isatidis Radix and Isatidis Folium. The harvest could increase the yield of Isatidis Folium, but reduce the yield of Isatidis Radix, the quality of Isatidis Radix and Isatidis Folium. One, two and three harvests of the Isatidis Folium reduced the yield of Isatidis Radix as 18.3%, 58.6%, 67.4% and increased the yield of the Folium as 107.3%, 86.3% and 116.6%. Ethanol-soluble extract of Isatidis Radix was 42.50%, 42.24%, 31.77%, which were 1.19%, 1.79%, 26.13% lower than those of the control, respectively. The water-soluble extract, indirubin, indigo content reduced with increase of the harvest times. Indirubin contents with two or three times harvests were higher than that of the control, but the content of water-soluble extract, ethanol-soluble extract, indigo were lower than those of the control.
Subject(s)
Agriculture , Methods , Desert Climate , Drugs, Chinese Herbal , Chemistry , Metabolism , Indigo Carmine , Indoles , Metabolism , Isatis , Metabolism , Plant Leaves , Chemistry , Metabolism , Plant Roots , Chemistry , Metabolism , Quality Control , Time FactorsABSTRACT
<p><b>OBJECTIVE</b>To explore an economical and practical storage method for angelica roots (Angelica sinensis). Various treatments were used in packaging and storing dry and fresh angelica roots.</p><p><b>METHOD</b>The experiment was designed in single-factor completely random as compared with the traditional stack without package. The character, weight, extract contents and volatile oil were measured within the storage process.</p><p><b>RESULT</b>If stored for long-term use, the dried angelica roots should be packaged with vacuum-exhausted bags cleaned with water, which resulted in less loss in weight, higher extracts and volatile oil. However, if stored for fresh use, the fresh angelica roots should be packaged in vacuum-exhausted bags after cleaned with sorghum alcohol and stored under low temperatures if possible, which resulted in less loss in weight and longer fresh time. Considering volatile oil and extract contents, it is better to use the dried angelica roots for storage.</p><p><b>CONCLUSION</b>If stored for long-term use the dry angelica roots should be packaged with vacuum-exhausted bags cleaned with water, while for fresh use the fresh angelica roots should be packaged with vacuum-exhausted bags cleaned with sorghum alcohol and stored under low temperatures if possible.</p>
Subject(s)
Angelica sinensis , Drug Packaging , Economics , Methods , Drug Storage , Economics , Methods , Drugs, Chinese Herbal , Plant Roots , Quality Control , Time FactorsABSTRACT
Objective The effect of fertilizer application on yield and quality of Notopterygium forbesii was studied in order to provide a theoretical basis for high quality and yield planting.Methods With various nitrogenous(N) and phosphorous(P) fertilizer formula,the field tests were carried out to determine the yield,extract content,and essential oil content,from the root of N.forbesill.Results Showed that various proportion of fertilizer had a great affect on yield,extract content,and essential oil content,and the treatment with N2P2 was the highest.The yield in the treatment with N2P2 increased 43.33%,the extract content increased 17.95%,and the essential oil content increased 43.48% as compared with CK.Different rates of fertilizer application had a certain effect on weight and diameter in the root of N.forbesii,and the N2P2 was the best.Each treatment had a significant effect on diameter in the root of N.forbesii in comparision with CK.Conclusion Amount and ratio of N and P fertilizers have a greater influence on the yield and quality,while improper application could reduce the yield and quality.