ABSTRACT
Objective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.. Methods The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers’ O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested. Results The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL. Conclusion The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.
ABSTRACT
Lao-Xiang-Huang (LXH) of Chaozhou is the processed product of Fructus Citri sarcodactylis.LXH from different producing areas were used as research objects in the study for the establishment of HPLC fingerprints of LXH.Contents of hesperidin and 5,7-dimethoxycoumarin were also determined to provide scientific basis for the establishment of quality standard of LXH.Samples were separated by an Agilent Eclipse XDB C18 column (4.6 mm × 250 mm,5 μm) using acetonitrile-0.1％ phosphoric acid with water gradient system as a mobile phase.The flow rate was 1.0 mL· min-1.The injection volume was 20 μL.The detection wavelength was at 283 nm.The results showed that HPLC fingerprint of LXH was established with good separation and repeatability.The similarity evaluation on 27 batches samples of LXH showed that there was a certain similarity on the HPLC fingerprints of LXH.However,there was a certain difference as a whole.Contents of hesperidin and 5,7-dimethoxycoumarin in LXH were simultaneously determined.It was concluded that the established HPLC fingerprint of LXH and content determination of hesperidin and 5,7-dimethoxycoumarin method were accurate,sensitive and repeatable.It provided scientific evidence for the quality control standard of LXH of Chaozhou.
ABSTRACT
Objective:To study the effect and underlying mechanism of sulfur-fumigation and water-soaking on total ash of Di-oscoreae Rhizoma, find the key factor( s) affecting the total ash of Dioscoreae Rhizoma, and explore the rationality of ash limits of Di-oscoreae Rhizoma described in Chinese Pharmacopoeia. Methods:Dioscoreae Rhizoma was respectively dealt with sulfur-fumigation and water-soaking. The changes in total ash content of Dioscoreae Rhizoma was detected by the ash determination methods for total ash and SO2 described in the pharmacopoeia, and then the ash content change of inorganic salts was used to study the mechanism. Results:Sulfur-fumigation could slightly reduce the total ash content of Dioscoreae Rhizoma, while significantly reduce the ash content of calcium oxalate and calcium sulfate with the reduction degree of 7. 20% and 9. 90%, respectively. Calcium phosphate and calcium chloride were slightly affected by sulfur-fumigation, and the results indicated that the effect of sulfur-fumigation on ash content was mainly real-ized by increasing the decomposition rate of calcium oxalate and calcium sulfate. Water-soaking could decline the ash content of Di-oscoreae Rhizoma, and the phenomenon was common in the rhizome medicinal materials. The influence of water-soaking on total ash was more significant than that of sulfur fumigation. Conclusion:Sulfur-fumigation can reduce the total ash content of Dioscoreae Rhizo-ma by increasing the decomposition rate of calcium oxalate and calcium sulfate, however, the effect is mild and the process isn't the key influencing factor in the total ash content of Dioscoreae Rhizoma. During the preparation of Dioscoreae Rhizoma medicinal slices, water-soaking can cause the great loss of water-soluble mineral salts, such as Cl-, C2 O4 2-, NO3 - and SO4 2-, which leads to the reduction of total ash content, therefore, water-soaking is the key influencing factor in the total ash content of Dioscoreae Rhizoma.
ABSTRACT
Sulfur , a major component of gunpowder , has been widely used in the engineering and military in-dustries since ancient times . In fact , the application of sulfur in traditional Chinese medicine ( TCM ) has a long history , which indicated the uniqueness of TCM theory and practice . Besides , sulfur has played an impor-tant role for the development of TCM in the history . In order to scientifically analyze the role of sulfur in TCM , this paper focused on the application and evolution of sulfur in the development process of TCM , which aimed to provide a reference for the study of the value and role of sulfur in TCM .