1.Phase Change and Quantity-quality Transfer Analysis of Medicinal Materials, Decoction Pieces and Standard Decoction of Haliotidis Concha (Haliotis discus hannai)
Zhihan YANG ; Jingwei ZHOU ; Weichao WANG ; Yu HUANG ; Chuang LUO ; Lian YANG ; Chenyu ZHONG ; Hongping CHEN ; Fu WANG ; Yuan HU ; Youping LIU ; Shilin CHEN ; Lin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(13):206-214
ObjectiveTo explore the quantity-quality transfer process of medicinal materials, decoction pieces and standard decoction of Haliotidis Concha(Haliotis discus hannai) by analyzing the physical phase and compositional changes, so as to provide references for the effective control of its quality. MethodsA total of 20 batches of Haliotidis Concha(H. discus hannai) from different habitats were collected and prepared into corresponding calcined products and standard decoction, and the content of CaCO3 of the three samples were determined and the extract yield and transfer rate of CaCO3 were calculated. The changes in elemental composition and their relative contents were investigated by X-ray fluorescence spectrometry(XRF), X-ray diffraction(XRD) was used to study the changes in the phase compositions of the three samples and to establish their respective XRD specific chromatogram. Fourier transform infrared spectrometry(FTIR) was used to study the changes in the chemical composition and content changes of the three samples and to establish their respective FTIR specific chromatogram, while combining hierarchical cluster analysis(HCA), principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) to find the common and differential characteristics, in order to explore the quantity-quality transfer relationship in the preparation process of standard decoction of Haliotidis Concha(H. discus hannai). ResultsThe CaCO3 contents of the 20 batches of medicinal materials, decoction pieces and standard decoction of Haliotidis Concha(H. discus hannai) were 93.87%-98.95%, 96.02%-99.97% and 38.29%-51.96%, respectively, and the extract yield of standard decoction was 1.71%-2.37%, and the CaCO3 transfer rate of decoction pieces-standard decoction was 0.68%-1.27%. XRF results showed that the elemental species and their relative contents contained in Haliotidis Concha and its calcined products had a high degree of similarity, and although there was no obvious difference in the elemental species contained in decoction pieces and standard decoction, the difference in the relative contents was obvious, which was mainly reflected in the decrease of the relative content of element Ca and the increase of the relative content of element Na. XRD results showed that Haliotidis Concha mainly contained CaCO3 of aragonite and calcite, while calcined Haliotidis Concha only contained CaCO3 of calcite, and standard decoction mainly contained CaCO3 of calcite and Na2CO3 of natrite. FTIR results showed that there were internal vibrations of O-H, C-H, C=O, HCO3- and CO32- groups in Haliotidis Concha, while O-H, HCO3- and CO32- groups existed in the calcined products and standard decoction. ConclusionThe changes of Haliotidis Concha and calcined Haliotidis Concha are mainly the increase of CaCO3 content, the transformation of CaCO3 aragonite crystal form to calcite crystal form and the absence of organic components after calcination, and the changes of calcined products and standard decoction are mainly the decrease of CaCO3 content and the increase of Na2CO3 relative content. The method established in the study is applicable to the quality control of the shellfish medicines-decoction pieces- standard decoction, which provides a new idea for the study of quality control of dispensing granules of shellfish medicines.
2.Phase Change and Quantity-quality Transfer Analysis of Medicinal Materials, Decoction Pieces and Standard Decoction of Haliotidis Concha (Haliotis discus hannai)
Zhihan YANG ; Jingwei ZHOU ; Weichao WANG ; Yu HUANG ; Chuang LUO ; Lian YANG ; Chenyu ZHONG ; Hongping CHEN ; Fu WANG ; Yuan HU ; Youping LIU ; Shilin CHEN ; Lin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(13):206-214
ObjectiveTo explore the quantity-quality transfer process of medicinal materials, decoction pieces and standard decoction of Haliotidis Concha(Haliotis discus hannai) by analyzing the physical phase and compositional changes, so as to provide references for the effective control of its quality. MethodsA total of 20 batches of Haliotidis Concha(H. discus hannai) from different habitats were collected and prepared into corresponding calcined products and standard decoction, and the content of CaCO3 of the three samples were determined and the extract yield and transfer rate of CaCO3 were calculated. The changes in elemental composition and their relative contents were investigated by X-ray fluorescence spectrometry(XRF), X-ray diffraction(XRD) was used to study the changes in the phase compositions of the three samples and to establish their respective XRD specific chromatogram. Fourier transform infrared spectrometry(FTIR) was used to study the changes in the chemical composition and content changes of the three samples and to establish their respective FTIR specific chromatogram, while combining hierarchical cluster analysis(HCA), principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) to find the common and differential characteristics, in order to explore the quantity-quality transfer relationship in the preparation process of standard decoction of Haliotidis Concha(H. discus hannai). ResultsThe CaCO3 contents of the 20 batches of medicinal materials, decoction pieces and standard decoction of Haliotidis Concha(H. discus hannai) were 93.87%-98.95%, 96.02%-99.97% and 38.29%-51.96%, respectively, and the extract yield of standard decoction was 1.71%-2.37%, and the CaCO3 transfer rate of decoction pieces-standard decoction was 0.68%-1.27%. XRF results showed that the elemental species and their relative contents contained in Haliotidis Concha and its calcined products had a high degree of similarity, and although there was no obvious difference in the elemental species contained in decoction pieces and standard decoction, the difference in the relative contents was obvious, which was mainly reflected in the decrease of the relative content of element Ca and the increase of the relative content of element Na. XRD results showed that Haliotidis Concha mainly contained CaCO3 of aragonite and calcite, while calcined Haliotidis Concha only contained CaCO3 of calcite, and standard decoction mainly contained CaCO3 of calcite and Na2CO3 of natrite. FTIR results showed that there were internal vibrations of O-H, C-H, C=O, HCO3- and CO32- groups in Haliotidis Concha, while O-H, HCO3- and CO32- groups existed in the calcined products and standard decoction. ConclusionThe changes of Haliotidis Concha and calcined Haliotidis Concha are mainly the increase of CaCO3 content, the transformation of CaCO3 aragonite crystal form to calcite crystal form and the absence of organic components after calcination, and the changes of calcined products and standard decoction are mainly the decrease of CaCO3 content and the increase of Na2CO3 relative content. The method established in the study is applicable to the quality control of the shellfish medicines-decoction pieces- standard decoction, which provides a new idea for the study of quality control of dispensing granules of shellfish medicines.
3.Potential Components and Mechanisms of Ganlu Xiaodu Dan in Treatment of Viral Pneumonia
Weichao ZHANG ; Yayun LI ; Tianci GAO ; Mengxing HOU ; Wenzhong XU ; Fenqiao CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):188-196
ObjectiveTo explore the mechanisms of action of Ganlu Xiaodu Dan in treating viral pneumonia by combining network pharmacology and molecular docking with in vivo experimental validation. MethodsNetwork pharmacology and molecular docking were used to predict the core components, target genes, and major pathways of Ganlu Xiaodu Dan. Molecular docking was then applied to verify the interactions between the core components and key targets. Sixty male C57BL/6 mice were randomly divided into six groups (n = 10 per group), including blank, model, dexamethasone, and Ganlu Xiaodu Dan low-, medium-, and high-dose groups. The blank and model groups were gavaged with physiological saline (10 mL·kg-1) every 12 h. The dexamethasone group received intraperitoneal injections of dexamethasone (5 mg·kg-1). The low-, medium-, and high-dose groups of Ganlu Xiaodu Dan were gavaged with solutions at concentrations of 7.2, 14.4, and 21.6 g·kg-1, respectively, every 12 h. Lung wet/dry weight ratio (W/D) was measured. Hematoxylin-eosin (HE) staining was used to observe pathological changes in lung tissue. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-17, and IL-1β in bronchoalveolar lavage fluid (BALF). Western blot was performed to detect the expression of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) in lung tissue for further validation. ResultsTwelve potential active components of Ganlu Xiaodu Dan were identified through network pharmacology. A total of 306 overlapping target genes were obtained between Ganlu Xiaodu Dan and viral pneumonia. PPI network analysis identified the top 20 key targets, and GO and KEGG enrichment analyses revealed the top 20 signaling pathways. An “active component–target–pathway” network was constructed. Molecular docking demonstrated strong affinity between the core components of Ganlu Xiaodu Dan and key targets related to viral pneumonia. In animal experiments, compared with the blank group, the model group showed severe bronchial epithelial damage, disordered alveolar structure, massive inflammatory cell infiltration, widened alveolar septa, and obvious interstitial edema. W/D, levels of IL-1β, TNF-α, and IL-17 in BALF, and protein expression of p-PI3K/PI3K and p-Akt/Akt in lung tissue were all significantly increased (P<0.05). Compared with the model group, lung injury in the Ganlu Xiaodu Dan groups and the dexamethasone group was alleviated. W/D and TNF-α levels were significantly decreased (P<0.05). IL-1β and IL-17 levels were significantly reduced in the medium- and high-dose groups and the dexamethasone group, and the protein expression levels of p-PI3K/PI3K and p-Akt/Akt in lung tissue were significantly decreased (P<0.05). ConclusionGanlu Xiaodu Dan can alleviate lung injury in viral pneumonia by suppressing the inflammatory response, and its mechanism may be related to the inhibition of PI3K/Akt pathway activation.
4.Potential Components and Mechanisms of Ganlu Xiaodu Dan in Treatment of Viral Pneumonia
Weichao ZHANG ; Yayun LI ; Tianci GAO ; Mengxing HOU ; Wenzhong XU ; Fenqiao CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):188-196
ObjectiveTo explore the mechanisms of action of Ganlu Xiaodu Dan in treating viral pneumonia by combining network pharmacology and molecular docking with in vivo experimental validation. MethodsNetwork pharmacology and molecular docking were used to predict the core components, target genes, and major pathways of Ganlu Xiaodu Dan. Molecular docking was then applied to verify the interactions between the core components and key targets. Sixty male C57BL/6 mice were randomly divided into six groups (n = 10 per group), including blank, model, dexamethasone, and Ganlu Xiaodu Dan low-, medium-, and high-dose groups. The blank and model groups were gavaged with physiological saline (10 mL·kg-1) every 12 h. The dexamethasone group received intraperitoneal injections of dexamethasone (5 mg·kg-1). The low-, medium-, and high-dose groups of Ganlu Xiaodu Dan were gavaged with solutions at concentrations of 7.2, 14.4, and 21.6 g·kg-1, respectively, every 12 h. Lung wet/dry weight ratio (W/D) was measured. Hematoxylin-eosin (HE) staining was used to observe pathological changes in lung tissue. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-17, and IL-1β in bronchoalveolar lavage fluid (BALF). Western blot was performed to detect the expression of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) in lung tissue for further validation. ResultsTwelve potential active components of Ganlu Xiaodu Dan were identified through network pharmacology. A total of 306 overlapping target genes were obtained between Ganlu Xiaodu Dan and viral pneumonia. PPI network analysis identified the top 20 key targets, and GO and KEGG enrichment analyses revealed the top 20 signaling pathways. An “active component–target–pathway” network was constructed. Molecular docking demonstrated strong affinity between the core components of Ganlu Xiaodu Dan and key targets related to viral pneumonia. In animal experiments, compared with the blank group, the model group showed severe bronchial epithelial damage, disordered alveolar structure, massive inflammatory cell infiltration, widened alveolar septa, and obvious interstitial edema. W/D, levels of IL-1β, TNF-α, and IL-17 in BALF, and protein expression of p-PI3K/PI3K and p-Akt/Akt in lung tissue were all significantly increased (P<0.05). Compared with the model group, lung injury in the Ganlu Xiaodu Dan groups and the dexamethasone group was alleviated. W/D and TNF-α levels were significantly decreased (P<0.05). IL-1β and IL-17 levels were significantly reduced in the medium- and high-dose groups and the dexamethasone group, and the protein expression levels of p-PI3K/PI3K and p-Akt/Akt in lung tissue were significantly decreased (P<0.05). ConclusionGanlu Xiaodu Dan can alleviate lung injury in viral pneumonia by suppressing the inflammatory response, and its mechanism may be related to the inhibition of PI3K/Akt pathway activation.
5.Construction and Thinking of Data Science System of Chronic Atrophic Gastritis
Jianhui SUN ; Weichao XU ; Xia ZHANG ; Runxue SUN ; Yanzhe CHEN ; Shaopo WANG ; Yuman WANG ; Zhen LIU ; Yanru DU ; Qian YANG ; Jianming JIANG
Journal of Traditional Chinese Medicine 2024;65(12):1208-1212
Taking chronic atrophic gastritis (CAG) as an example, the frontier technologies in data science have been introduced into the inheritance, innovation and development of traditional Chinese medicine (TCM), providing reference for conducting real-world clinical research on specialized diseases of TCM. This paper put forward the construction of CAG data science system by elaborating the connotation of data science and its application value in TCM, and discussed the path to build CAG data science system, namely through "data acquisition-knowledge expression-knowledge reasoning" to establish CAG database, knowledge base and develop diagnosis platform differentiating diseases and syndromes. Besides, this paper analyzed the prospects of CAG data science in improving data governance ability and knowledge discovery efficiency, deepening the level of knowledge sharing, promoting interdisciplinary integration, and strengthening the integration process of industry, academia and research.
6.Correlation analysis on occupational acid fog exposure and accelerated biological aging in workers
Weichao WU ; Yan GUO ; Xiangkai ZHAO ; Zhiguang GU ; Yijia GUO ; Zipeng LAN ; Hui HUANG ; Lei KUANG ; Ming ZHANG ; Dongsheng HU ; Yongli YANG ; Wei WANG ; Jinru CHEN
Journal of Jilin University(Medicine Edition) 2024;50(6):1741-1750
Objective:To discuss the association between occupational acid fog exposure and accelerated biological aging of the workers,and to clarify its related risk factors.Methods:A total of 341 male workers exposed to occupational acid fog and 201 male workers without occupational exposure were selected as the study subjects,and they were divided into exposure group and control group,respectively.The general informations of the subjects in two groups were collected through questionnaires and physical examinations.The levels of red blood cell count(RBC),platelet count(PLT),albumin(ALB),urea(Urea),creatinine(CR),triglycerides(TG),total cholesterol(TC),glycated hemoglobin(HBA1c),and high-sensitivity C-reactive protein(Hs-CRP)in serum of the subjects in two groups were detected.The Klemera-Doubal method(KDM)was used to construct the composite aging measure,KDM-biological age(BA)(KDM-BA).The model parameters were trained using samples from the 2009 China Health and Nutrition Survey(CHNS)Database to calculate the BA acceleration of the subjects in two groups;stratified analysis based on the population characteristics was conducted to analyze the BA of the subjects in two groups with different population characteristics;generalized linear model was used to analyze the factors influencing BA acceleration due to acid fog exposure.Results:The model parameters were trained using samples from the 2009 CHNS Database,including 8 133 cases aged 20-79 years,of which 3 788 were male.The levels of Urea,CR,HBA1c,ALB,and TC,as well as systolic blood pressure(SBP),total working years,sleep duration,and body mass index(BMI)of the subjects between two groups had significant differences(P<0.05).Compared with control group,the BA acceleration of the subjects in exposure group was significantly increased(P<0.05).In entire population and exposure group,the BA acceleration in the smokers was significantly higher than that in the non-smokers(P<0.05).In entire population,control group,and exposure group,the BA accelerations of the subjects in different BMI groups were significantly decreased with the increase of BMI(P<0.05).Compared with control group,the BA acceleration of the subjects in exposure group was significantly increased(P<0.05),including those under 40 years old,with total working years of 4-7 years,Han nationality,unmarried,smokers,and sleep duration 6-7 h,and with overweight.Acid fog exposure,smoking,and BMI were associated with the BA acceleration(β=0.72,95%CI:0.24-1.21;β=0.59,95%CI:0.11-1.06;β=-0.29,95%CI:-0.35—-0.22).Conclusion:Occupational acid fog exposure may accelerate the biological aging in the workers,and acid fog is a risk factor to accelerate the biological aging of the body.
7.Mechanism of miR-15a-5p regulation of Wnt pathway in paraquat-induced pulmonary fibrosis
Jing WANG ; Xiaohang JI ; Mengmeng WANG ; Wei ZHANG ; Weichao DING ; Juan CHEN ; Jing FENG ; Jiankang MENG ; Zhaorui SUN ; Shinan NIE
Chinese Journal of Emergency Medicine 2024;33(8):1128-1133
Objective:To investigate the effect and molecular mechanism of miR-15a-5p regulation Wnt signaling pathway in PQ-induced pulmonary fibrosis.Methods:The PQ-induced 16HBE cell model was constructed, high-throughput miRNA chip and RT-qPCR were used to screen for miR-15a-5p with significant differences. The experimental groups were as follows: NC group (normal control);no special treatment; PQ group: 50 μmol/L PQ treated cells for 72 h; miR-15a-5p group: 16HBE stable cell lines transfected with miR-15a-5p overexpressing lentivirus; miR-15a-5p+PQ group: Stable cell lines were treated with 50 μmol/L PQ for 72 h. The expression of Wnt pathway-related genes Wnt3α and β-catenin, fibroblast marker genes Collagen I, Vimentin and α SMA, epithelial marker genes Occludin and CK18 were detected by RT-qPCR and Western blot. The mice model of PQ-induced pulmonary fibrosis was constructed, and the protein expression and lung tissue injury were detected by Western blot, HE staining and immunohistochemistry. Data were expressed as mean ± standard deviation, and independent sample t-test was used to analyze the data between the two groups. Results:The expressions of Wnt3α, β-catenin, fibroblast marker genes Collagen I, Vimentin and α SMA significantly up-regulated in cell injury models ( P<0.05), the epithelial cell marker genes Occludin and CK18 significantly down-regulated ( P<0.05), overexpression of miR-15a-5p could inhibit the expression of Wnt3α and alleviated the EMT induced by PQ. In animal models, Wnt3α, β-catenin, fibroblast marker genes Collagen I, Vimentin and α SMA significantly increased ( P<0.01), the structure of lung tissue was disordered and fibrosis occurred, overexpression of miR-15a-5p inhibited the expression of Wnt3α protein ( P<0.05) and ameliorated lung tissue injury. Conclusions:miR-15a-5p ameliorates PQ-induced lung injury by modulating the Wnt3α/β-catenin signaling pathway, thereby inhibiting the development of pulmonary fibrosis.
8.Protective effect of Xuebijing injection on sepsis-associated acute respiratory distress syndrome by suppressing the HIF-1α/p38 MAPK/NF-κB signaling pathway
Weichao DING ; Juan CHEN ; Xiaohang JI ; Yi REN ; Wei ZHANG ; Mengmeng WANG ; Jing FENG ; Xinyao WU ; Jiankang MENG ; Shinan NIE ; Zhaorui SUN
Chinese Journal of Emergency Medicine 2024;33(8):1140-1150
Objective:To explore the protective mechanism of Xuebijing injection (referred to as Xuebijing) on sepsis-associated acute respiratory distress syndrome (ARDS).Methods:① Animal experiments: 100 mice were randomly(random number) divided into 4 groups, including sham operation (Sham) group, cecal ligation and puncture (CLP) group, CLP+low-dose Xuebijing (L-XBJ) group, and CLP+high-dose Xuebijing (H-XBJ) group. The survival rate, lung histological changes, lung wet/dry (W/D) ratio, cell count and protein concentration in bronchoalveolar lavage fluids (BALF), inflammatory factors levels in serum, oxidative stress indicators, cell apoptosis, and key proteins of HIF-1α/p38 MAPK/NF-κB signaling pathway were measured. ② Cell experiments: Mouse alveolar macrophages (MH-S) were cultured in vitro and divided into 6 groups, including control (Con) group, lipopolysaccharide (LPS) group, LPS+L-XBJ group, and LPS+H-XBJ group, LPS+H-XBJ+ dimethyloxallyl glycine (DMOG, HIF-1α activator) group, LPS+H-XBJ+ 2-methoxyestradiol (2ME2, HIF-1α inhibitor) group. The effects of Xuebijing on inflammatory factors, oxidative stress, and cell apoptosis and their relationship with HIF-1α/p38 MAPK/NF-κB signaling pathway were detected.Results:Xuebijing increased the survival rate of mice with sepsis-associated ARDS, relieved lung tissue damage [lung injury score: CLP group (8.778±0.588), CLP+L-XBJ group (5.833±0.310), and CLP+H-XBJ group (4.750±0.246)], alleviated lung W/D ratio, and decreased pneumonia cell infiltration and protein exudation (all P<0.05). Additionally, Xuebijing treatment also diminished the expression of inflammatory factors (TNF-α, IL-1β, and IL-6), intracellular reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) formation, superoxide dismutase (SOD) depletion, and cell apoptosis in LPS-induced MH-S cells and CLP-induced sepsis-associated ARDS mice (all P<0.05). Furthermore, mechanistic investigation further clarified the effects of Xuebijing on inflammation, oxidative stress, and cell apoptosis through the HIF-1α/p38 MAPK/NF-κB signaling pathway. Conclusions:Xuebijing can exert anti-inflammatory, anti-oxidative, and anti-apoptotic effects by suppressing the HIF-1α/p38 MAPK/NF-κB signaling pathway, thereby conferring protection against sepsis-associated ARDS.
9.Comparative analysis of CT features of gastrointestinal stromal tumors,neurogenic tumors and leiomyomas in stomach
Yue LI ; Guihan YANG ; Weichao YANG ; Fengyan CHENG ; Yulin LIN ; Chun YAO ; Xiaofeng CHEN ; Zhiqi YANG
Journal of Practical Radiology 2024;40(8):1296-1299,1333
Objective To investigate the difference of CT features of gastrointestinal stromal tumors(GIST),neurogenic tumors,and leiomyomas in stomach.Methods A retrospective analysis was performed on clinical and CT features from 312 cases of GIST,21 cases of neurogenic tumors,and 35 cases of leiomyomas in stomach.Results GIST were most commonly found in the body of stomach and exhibited large tumor sizes and late onset.CT showed GIST predominantly showed intraluminal and mixed growth pattern with irregular or round shapes and uneven density,and cystic degeneration and calcification were frequently observed.The CT values of GIST in the arterial phase and the degree of arterial enhancement were higher,with light to moderate arterial phase enhancement and moderate to marked venous phase enhancement.Gastric leiomyomas had smaller tumor sizes and presented mainly in the cardia,gastric fundus,and lesser curvature of gastric body.CT showed the intraluminal growth pattern,primarily in round shapes with uniform density,lower incidence of cystic degeneration and calcification,both the arterial and venous phase CT values and the extent of enhancement in these phases were lower,showed no or slight enhancement during the arterial phase and light to moderate enhancement during the venous phase.Gastric neurogenic tumors were predominantly located in the gastric body and antrum.CT showed the tumors demonstrated extraluminal and mixed growth patterns,most with oval-shaped appearace,uniform density and the venous phase CT values and the degree of enhancement were higher,with light to moderate arterial enhancement and moderate to marked venous enhancement.Conclusion GIST,neurogenic tumors,and leiomyomas in stomach can be differentiated based on their distinct CT features.Accurate recognition of these features aids in the differential diagnosis of these kinds of tumors.
10.Rapid diagnosis of invasive candidiasis by droplet digital PCR
Zhijie HE ; Weichao LI ; Minghui HE ; Xiaotong CHEN ; Zhao LIN ; Yaowei ZHI
The Journal of Practical Medicine 2024;40(19):2738-2746
Objective To establish a rapid detection method for invasive candidiasis based on droplet digital polymerase chain reaction(ddPCR).Methods We developed an assay system using a microtitre-based digital PCR platform and designed primer probes specific for four Candida species,namely Candida albicans,Candida smoothii,Candida near-smoothii,and Candida tropicalis.(1)The Limit of Blank(LOB)range and positive judg-ment value were determined by analyzing No Template Control(NTC)samples.(2)The Limit of Detection(LOD)range was determined by diluting positive samples with 10 replicate extractions at each concentration gradient.(3)The Linear Limit of Quantitation(LOQ)range was determined by repetitive testing of diluted samples.(4)The linear range limit was determined through gradient dilution of the positive samples.(5)The coefficient of variation(CV),calculated from the logarithmic values of the resultant concentrations,was assessed by extracting and test-ing positive samples in 12 repetitions at both high and low concentrations.(6)Method reliability was evaluated by calculating the CV from the logarithmic values of the resultant concentrations obtained from clinical samples with fungal culture results.Results The ddPCR assay detected Candida LOB at a range of 0~81 copies/mL,with a positive threshold set at≥3 positive microdroplets.The LOD and LOQ were determined to be 3×102 copies/mL.The linear range for detecting different concentration gradients was found to be between 3×102 and 3×107 copies/mL,with high correlation coefficients observed for Candida albicans(R2=0.999 5),Candida smoothii(R2=0.998 9),Candida near-smoothii(R2=0.999 4),and Candida tropicalis(R2=0.999).Additionally,the coefficient of variation for the resultant concentration logarithmic values was less than 5%,meeting precision requirements.Furthermore,preliminary validation using clinical specimens demonstrated consistent results compared to clinical culture findings.Conclusion ddPCR exhibits rapidity,high sensitivity,good repeatability,and high specificity in detecting inva-sive candidiasis in critically ill patients.This study highlights the potential value of droplet digital PCR as a diag-nostic tool for invasive candidiasis.

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