1.Shenqi Yiliu Prescription Reverses Cisplatin Resistance in Ovarian Cancer Cells by Regulating PI3K/Akt/mTOR Signaling Pathway-mediated Glycolysis
Lan MA ; Yuping YANG ; Min BAI ; Yongqiang DUAN ; Zhining ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):60-69
ObjectiveTo investigate the mechanism by which Shenqi Yiliu prescription reverses cisplatin resistance in ovarian cancer cells by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway-mediated glycolysis. MethodsThe human ovarian cancer A2780 cell line was intervened with progressively increasing doses of cisplatin (1 g·L-1) to establish the cisplatin-resistant cell line A2780cisR, and the cell sensitivity to cisplatin was examined by the cell counting kit-8 (CCK-8) assay. High, medium, and low (39.9, 19.95, 9.98 g·kg-1) doses of Shenqi Yiliu prescription-containing sera were used to treat A2780cisR cells for 48 h. Glucose consumption and lactate production were measured by the cuvette assay. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the activities of glucose transporter (GLUT), phosphofructokinase (PFK), and pyruvate kinase (PK). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptosis. Western blot was employed to quantify the protein levels of phosphorylated (p)-PI3K, p-Akt, p-mTOR, hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), B-cell lymphoblastoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax), and B-lymphoblastoma-2 gene-related promoter (Bad). Real-time PCR was conducted to determine the mRNA levels of HK2, PKM2, Bax, Bcl-2, and Bad. ResultsThe median inhibitory concentration (IC50) of cisplatin on A2780cisR cells was nearly 3 times that on A2780P cells. Compared with A2780P cells, A2780cisR cells showed increased glucose consumption, lactate production, GLUT, PFK, and PK activities, and mRNA and protein levels of p-PI3K, Akt, p-mTOR, HK2, PKM2, Bax (P<0.05), and decreased apoptosis rate and Bcl-2 expression (P<0.05). Compared with A2780cisR cells, medium- and high-dose Shenqi Yiliu prescription reduced the glucose consumption, lactate production, GLUT, PFK, and PK activities, and mRNA and protein levels of p-PI3K, Akt, p-mTOR, HK2, PKM2, Bax, and Bad (P<0.05), while increasing the apoptosis rate and Bcl-2 expression (P<0.05). ConclusionShenqi Yiliu prescription can inhibit glycolysis mediated by the PI3K/Akt/mTOR pathway to promote apoptosis, thereby reversing cisplatin resistance in ovarian cancer cells.
2.Efficacy and Application Characteristics of Cold Chinese Medicines Based on Chinese Pharmacopoeia (2020 Edition)
Lu YUE ; Yilong HU ; Jingying YANG ; Xiangxiang WU ; Mingsan MIAO ; Ming BAI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):241-248
ObjectiveTo provide a reference for the rational clinical use of cold Chinese medicines by sorting and analyzing their properties, flavors, meridian tropism, primary therapeutic indications, methods of administration, dosages, and precautions as recorded in the 2020 edition of Pharmacopoeia of the People's Republic of China (Chinese Pharmacopoeia). MethodsCold Chinese medicines for internal and external use included in the 2020 edition of Chinese Pharmacopoeia were entered one by one, and their efficacy, properties, flavors, meridian tropism, methods of administration, dosages, and usage precautions were statistically classified and summarized to guide clinical medication use. ResultsA total of 259 cold Chinese medicines for internal use were included and categorized into 18 efficacy groups, mainly comprising heat-clearing drugs, water-excreting and dampness-draining drugs, and phlegm-resolving, cough- and asthma-relieving drugs. Their predominant flavors were bitter, sweet, and pungent, and they primarily entered the liver, lung, and stomach meridians. The main methods of administration included decocting first, grinding into powder for oral use, or preparing into pills or powders, with most dosages ranging from 9 to 15 g. A total of 83 cold Chinese medicines for external use were included, involving 16 efficacy categories. Their main flavors were bitter, sweet, and pungent, primarily entering the liver, lung, and large intestine meridians. The main external application methods were grinding into powder for topical use or preparing decoctions for fumigation and washing, with most dosages ranging from 9 to 15 g. Whether for internal or external use, cold Chinese medicines should be used with caution or contraindicated in pregnant women. ConclusionThe cold Chinese medicines included in the 2020 edition of the Chinese Pharmacopoeia are mainly suitable for patients with carbuncles, swellings, and coughs. However, in clinical practice, it is necessary to strictly follow the principles of syndrome differentiation and treatment, pay attention to administration methods and dosages, and use cold medicines rationally and effectively to improve clinical efficacy.
3.Current Status and Prospects of Research on Traditional Chinese Medicine Prevention and Treatment for Gastric Precancerous Lesions
Haiyan BAI ; Tai ZHANG ; Ping WANG ; Lin LIU ; Weichao XU ; Yaxin TIAN ; Lanshuo HU ; Qian YANG ; Xudong TANG
Journal of Traditional Chinese Medicine 2026;67(4):410-415
Traditional Chinese medicine (TCM), through its multi-target and systematic regulatory effects, has demonstrated unique advantages in the treatment of gastric precancerous lesions (GPL). At present, TCM theoretical research on GPL is mainly reflected in three aspects, the integration of macroscopic syndrome differentiation, the inflammation-carcinoma transformation mechanism, as well as the systematization and scientization of theoretical inheritance from famous TCM practitioners. High-quality evidence-based research findings serve as the foundation for clinical practice guidelines on GPL, and TCM has gained international academic recognition in the field of GPL prevention and treatment. Research on TCM mechanisms has yielded a series of important outcomes in the aspects of signaling pathways, gene expression regulation, cellular epigenetics, histone modification, and intestinal microecology. It is proposed that future research on GPL should focus on four key directions, establishing multi-omics data, exploring targeted intervention strategies on key regulatory nodes, advancing the standardization process of integrated traditional Chinese and western medicine prevention and treatment technologies, and constructing stratified screening and intervention platforms. The in-depth integration of TCM microcosmic mechanism of action with its macroscopic syndrome differentiation and treatment system, coupled with interdisciplinary research, will provide valuable references for the clinical treatment and scientific research of GPL.
4.Clinical Study on Treatment of Melasma Using Angelica and Safflower Spot Removal Essence Based on State-target Medicine
Bailin CHEN ; Haoyu YANG ; Rui CAI ; Yanping BAI ; Yuanyuan CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(2):111-119
ObjectiveTo investigate the clinical efficacy and safety of state-target formula Angelica and Safflower spot removal essence in the treatment of melasma. MethodsA randomized,double-blind,placebo-controlled parallel-group trial was conducted,selecting patients with Qi stagnation and blood stasis type of melasma who visited the dermatology outpatient department of Beijing Hospital of Integrated Traditional Chinese and Western Medicine from September 2022 to February 2023. In accordance with the double-blind principle,a random number table was generated to randomly divide the patients into an experimental group (50 cases) and a control group (50 cases). The experimental group applied the Angelica and Safflower spot removal essence topically,while the control group applied a placebo topically,with continuous treatment for 8 weeks and a follow-up of 1 month. Melasma Area and Severity Index (MASI),Physician Global Assessment (PGA),patient self-assessment scoring,VISIA detection and scoring were performed before medication and on the first day after stopping medication,and all adverse events were recorded. ResultsIn a study involving 100 patients,87 cases were included in the analysis. The experimental group consisted of 45 cases,with 5 dropouts,and the control group had 42 cases,with 8 dropouts. The baseline data of the patients in both groups were consistent,with no statistically significant differences. After treatment,the total effective rate of the experimental group was 75.56% (34/45),significantly higher than the control group's 2.38% (1/42),and the difference was statistically significant (χ2=48.38,P<0.01). Compared with before treatment,after treatment,the MASI score,VISIA image spot,brown spot,and red area score of the experimental group patients were significantly reduced (P<0.05,P<0.01),while there was no statistically significant difference in the control group patients. Compared with the control group after treatment,the experimental group showed more significant improvement (P<0.05,P<0.01). VISIA image analysis showed that after 8 weeks of treatment,the experimental group showed significant improvement in surface spots,deep spots,and red areas,while the control group remained unchanged or showed an increasing trend. 42.22% (19/45) of the experimental group had a PGA score of 3 or below,while 88.89% (40/45) had a PGA score of 4 or below. 80.95% (34/42) of the control group had a PGA score of 5 or above,and the difference between the two groups was statistically significant (χ2=38.26,P<0.01). There was no statistically significant difference in the recurrence rate and adverse reaction rate within one month between the experimental group and the control group. ConclusionThe state-target formula Angelica and Safflower spot removal essence is clinically effective and safe in the treatment of melasma.
5.Qingre Sanzhuo Decoction Treats Gouty Arthritis Combined with Hyperuricaemia in Rats via NLRP3/ASC/Caspase-1 Pathway
Haolin LI ; Qian BAI ; Weigang CHENG ; Weiqing LI ; Juanjuan YANG ; Peixin HE ; Huijun YANG ; Haidong WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):49-57
ObjectiveTo investigate the effect and mechanism of Qingre Sanzhuo decoction in treating gouty arthritis (GA) combined with hyperuricaemia (HUA). MethodsSixty male SD rats were randomized into normal, model, colchicine (0.5 mg·kg-1), and low-, medium-, and high-dose (17, 34, 68 g·kg-1, respectively) Qingre Sanzhuo decoction groups (n=10). The rats in other groups except the normal group were treated with the modified method for the modeling of GA combined with HUA. The drug intervention groups were administrated with corresponding drugs by gavage in the afternoon every day and the normal group and the model group were administrated with an equal volume of sterile normal saline by gavage. The level of uric acid (SUA) in the serum was measured 2 h after the last administration. The degree of ankle joint swelling was calculated 0.5, 12, 24, 48 h after modeling, and joint inflammation was scored. The pathological changes of ankle joints were observed by hematoxylin-eosin staining, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), C reactive protein (CRP), and interleukin-18 (IL-18) were measured by enzyme-linked immunosorbent assay. Real-time PCR was performed to determine the mRNA levels of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), cysteinyl aspartate-specific protease-1 (Caspase-1), gasdermin D (GSDMD), and nuclear factor-kappa B (NF-κB) in the synovial tissue of ankle joints. Western blot was employed to determine the protein levels of NLRP3, ASC, and Caspase-1 in ankle joints. The immunohistochemical method was used to detect the expression of GSDMD and NF-κB in the synovial tissue of ankle joints. ResultsCompared with the normal group, the model group showed increased SUA in the serum (P<0.05), ankle joint swelling and joint inflammation (P<0.05), increased number of blood vessels in the synovium, inflammatory cell foci in the synovial bursa, elevated serum levels of TNF-α, IL-1β, CRP, and IL-18 (P<0.05), and up-regulated mRNA and protein levels of NLRP3, ASC, Caspase-1, GSDMD, and NF-κB in the synovial tissue of ankle joints (P<0.05). Compared with the model group, the medium- and high-dose Qingre Sanzhuo decoction groups showed reduced SUA in the serum (P<0.05), alleviated ankle joint swelling and joint inflammation (P<0.05), lowered serum levels of TNF-α, IL-1β, CRP, and IL-18 (P<0.05), and down-regulated mRNA and protein levels of NLRP3, ASC, Caspase-1, GSDMD, and NF-κB in the synovial tissue of ankle joints (P<0.05). However, in terms of ameliorating the pathological changes of ankle joints, only the high-dose Qingre Sanzhuo decoction group showed normal morphology of the synovial membrane of ankle joints and no obvious lesion in the articular cartilage. ConclusionQingre Sanzhuo decoction may play a role in preventing and controlling GA combined with HUA by down-regulating the activity of NLRP3/ASC/Caspase-1 pathway and inhibiting the expression of inflammatory cytokines, such as TNF-α, IL-1β, CRP, and IL-18.
6.Construction of a Disease-Syndrome Integrated Diagnosis and Treatment System for Gastric "Inflammation-Cancer" Transformation Based on Multi-Modal Phenotypic Modeling
Hao LI ; Huiyao ZHANG ; Wei BAI ; Tingting ZHOU ; Guodong HUANG ; Xianjun RAO ; Yang YANG ; Lijun BAI ; Wei WEI
Journal of Traditional Chinese Medicine 2025;66(5):458-463
By analyzing the current application of multi-modal data in the diagnosis of gastric "inflammation-cancer" transformation, this study explored the feasibility and strategies for constructing a disease-syndrome integrated diagnosis and treatment system. Based on traditional Chinese medicine (TCM) phenomics, we proposed utilizing multi-modal data from literature research, cross-sectional studies, and cohort follow-ups, combined with artificial intelligence technology, to establish a multi-dimensional diagnostic and treatment index system. This approach aims to uncover the complex pathogenesis and transformation patterns of gastric "inflammation-cancer" progression. Additionally, by dynamically collecting TCM four-diagnostic information and modern medical diagnostic information through a long-term follow-up system, we developed three major modules including information extraction, multi-modal phenotypic modeling, and information output, to make it enable real-world clinical data-driven long-term follow-up and treatment of chronic atrophic gastritis. This system can provide technical support for clinical diagnosis, treatment evaluation, and research, while also offering insights and methods for intelligent TCM diagnosis.
7.Clinical Study on the Treatment of 70 Cases Chronic Atrophic Gastritis with Intestinal Metaplasia Using Xianglian Huazhuo Granules (香连化浊颗粒):A Randomized,Double-Blind,Placebo-Controlled Trial
Ziyu LI ; Maopeng ZHANG ; Wen ZHAO ; Wei LI ; Shiyun SHENG ; Haiyan BAI ; Qian YANG
Journal of Traditional Chinese Medicine 2025;66(5):473-479
ObjectiveTo observe the clinical efficacy and possible mechanisms of Xianglian Huazhuo Granules (香连化浊颗粒, XHG) in the treatment of chronic atrophic gastritis with intestinal metaplasia. MethodsA total of 140 patients with chronic atrophic gastritis and intestinal metaplasia were randomly divided into a treatment group and a control group, with 70 cases in each group. The treatment group received 12.5 g of XHG orally, twice daily. The control group received 12.5 g of placebo orally, twice daily. Both groups were treated for 6 months. The traditional Chinese medicine (TCM) symptom scores, pathological types, serum tumor markers of the digestive system, and serum bile acids (TBA), interleukin-23 (IL-23), and Dickkopf-related protein 1 (DKK-1) levels were observed before and after treatment. Safety indicators and adverse events were recorded. After treatment, TCM syndrome efficacy and pathological types were evaluated, and patients were followed up for 18 months with gastric endoscopy and pathological results, which were compared with the results after treatment finished. ResultsTwo patients dropped out in the control group, and a total of 168 cases were included in the final analysis, 70 in the treatment group and 68 in the control group. The treatment group showed a significant reduction in TCM symptom scores, serum TBA, IL-23, and DKK-1 levels, and a significant increase in alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 199 (CA199) levels; in the control group, carcinoembryonic antigen (CEA), CA125, CA199 levels significantly increased (P<0.05 or P<0.01); and carbohydrate antigen 242 (CA242) level in both the treatment group and the control group decreased significantly (P<0.01). The treatment group had lower TCM symptom scores and lower levels of serum TBA, IL-23, and DKK-1 compared to the control group (P<0.05). The effective rate for TCM syndrome efficacy in the treatment group was 80.00% (56/70), significantly higher than the 20.59% (14/68) in the control group (P < 0.05). The effective rate for pathological classification in the treatment group was 72.73% (8/11) for mixed intestinal metaplasia, significantly better than 46.15% (6/13) in the control group (P<0.05). No adverse events were reported in either group. Among 40 patients who had a follow-up endoscopy after one year, 21 were from the treatment group, of whom 11 showed reduced intestinal metaplasia, 9 showed no significant changes, and 1 had worsened; while 19 patients in the control group had 4 with reduced intestinal metaplasia, 13 with no significant changes, and 2 with worsened conditions. No cancer was detected in either group. The treatment group showed significantly better improvement in intestinal metaplasia on follow-up gastric endoscopy pathology than the control group (P<0.05). ConclusionXHG can significantly improve the clinical symptoms in patients with chronic atrophic gastritis and intestinal metaplasia and reduce the degree of mixed intestinal metaplasia. The mechanism may involve lowering serum TBA, DKK-1, and IL-23 levles, thus delaying the progression from inflammation to cancer.
8.Effects of chronic exposure to low-frequency pulsed magnetic fields on contractility and morphology of the quadriceps muscle in healthy adults
Xuanqiang WANG ; Wenyang ZHANG ; Yang LI ; Weiqian KONG ; Wei LI ; Le WANG ; Zhongshan LI ; Shi BAI
Chinese Journal of Tissue Engineering Research 2025;29(8):1634-1642
BACKGROUND:Changes in skeletal muscle mass have been indicated in studies addressing the effects of low-frequency pulsed magnetic fields on the structure and morphology of the skeletal muscle,but no relevant studies have been conducted on the morphologic changes that occur after chronic exposure to the low-frequency pulsed magnetic field. OBJECTIVE:To observe the effects of chronic exposure to low-frequency pulsed magnetic fields on the maximal voluntary contraction and morphologic indicators of the quadriceps muscle of the leg,thereby providing a reference of muscle morphologic changes for the use of this technique as a strategy for muscle function improvement. METHODS:Seventy healthy subjects were recruited and randomly divided into a test group that received magnetic field stimulation and a control group that underwent sham treatment,with 35 subjects in each group,and the total duration of the trial was 4 weeks.The test group underwent low-frequency pulsed magnetic stimulation for 15 minutes every 48 hours,while the control group underwent sham treatment,with the same intervention interval and duration as the test group.After 4 weeks of intervention,changes in the maximum voluntary contraction value of the quadriceps muscle in different groups were observed,and B-mode ultrasonography was utilized as a means of assessment to observe changes in muscle thickness,muscle cross-sectional area,and pinnation angle indexes. RESULTS AND CONCLUSION:After 4 weeks of chronic exposure to low-frequency pulsed magnetic fields,68 subjects completed the test.The maximum voluntary contraction value of the quadriceps muscle in the test group increased significantly(P=0.000),and the increment was significantly higher than that of the control group(P=0.008).Three indexes related to muscle morphology in the test group were significantly higher than the pre-test values(P=0.000),while in the control group,muscle thickness showed a significant reduction(P=0.020),there was no significant change in the pinnation angle,but a significant increase in the cross-sectional area(P=0.000).Intergroup comparisons revealed that the three indicators related to muscle morphology,including muscle thickness(P=0.012),pinnation angle(P=0.003),and cross-sectional area(P=0.049),were significantly higher in the test group than in the control group.The above data confirmed that the maximum voluntary contraction of the quadriceps muscle was significantly increased in healthy adults after 4 weeks of chronic exposure to the low-frequency pulsed magnetic field,and significant increases in the three muscle morphometric indices of muscle thickness,cross-sectional area,and pinnation angle were observed in the test group,providing a basis of muscle tissue morphology for the use of this technique as an exercise alternative and medical treatment strategy for muscle improvement.
9.Evaluation of the performance of the artificial intelligence - enabled snail identification system for recognition of Oncomelania hupensis robertsoni and Tricula
Jihua ZHOU ; Shaowen BAI ; Liang SHI ; Jianfeng ZHANG ; Chunhong DU ; Jing SONG ; Zongya ZHANG ; Jiaqi YAN ; Andong WU ; Yi DONG ; Kun YANG
Chinese Journal of Schistosomiasis Control 2025;37(1):55-60
Objective To evaluate the performance of the artificial intelligence (AI)-enabled snail identification system for recognition of Oncomelania hupensis robertsoni and Tricula in schistosomiasis-endemic areas of Yunnan Province. Methods Fifty O. hupensis robertsoni and 50 Tricula samples were collected from Yongbei Township, Yongsheng County, Lijiang City, a schistosomiasis-endemic area in Yunnan Province in May 2024. A total of 100 snail sample images were captured with smartphones, including front-view images of 25 O. hupensis robertsoni and 25 Tricula samples (upward shell opening) and back-view images of 25 O. hupensis robertsoni and 25 Tricula samples (downward shell opening). Snail samples were identified as O. hupensis robertsoni or Tricula by schistosomiasis control experts with a deputy senior professional title and above according to image quality and morphological characteristics. A standard dataset for snail image classification was created, and served as a gold standard for recognition of snail samples. A total of 100 snail sample images were recognized with the AI-enabled intelligent snail identification system based on a WeChat mini program in smartphones. Schistosomiasis control professionals were randomly sampled from stations of schistosomisis prevention and control and centers for disease control and prevention in 18 schistosomiasis-endemic counties (districts, cities) of Yunnan Province, for artificial identification of 100 snail sample images. All professionals are assigned to two groups according the median years of snail survey experiences, and the effect of years of snail survey experiences on O. hupensis robertsoni sample image recognition was evaluated. A receiver operating characteristic (ROC) curve was plotted, and the sensitivity, specificity, accuracy, Youden’s index and the area under the curve (AUC) of the AI-enabled intelligent snail identification system and artificial identification were calculated for recognition of snail sample images. The snail sample image recognition results of AI-enabled intelligent snail identification system and artificial identification were compared with the gold standard, and the internal consistency of artificial identification results was evaluated with the Cronbach’s coefficient alpha. Results A total of 54 schistosomiasis control professionals were sampled for artificial identification of snail sample image recognition, with a response rate of 100% (54/54), and the accuracy, sensitivity, specificity, Youden’s index, and AUC of artificial identification were 90%, 86%, 94%, 0.80 and 0.90 for recognition of snail sample images, respectively. The overall Cronbach’s coefficient alpha of artificial identification was 0.768 for recognition of snail sample images, and the Cronbach’s coefficient alpha was 0.916 for recognition of O. hupensis robertsoni snail sample images and 0.925 for recognition of Tricula snail sample images. The overall accuracy of artificial identification was 90% for recognition of snail sample images, and there was no significant difference in the accuracy of artificial identification for recognition of O. hupensis robertsoni (86%) and Tricula snail sample images (94%) (χ2 = 1.778, P > 0.05). There was no significant difference in the accuracy of artificial identification for recognition of snail sample images with upward (88%) and downward shell openings (92%) (χ2 = 0.444, P > 0.05), and there was a significant difference in the accuracy of artificial identification for recognition of snail sample images between schistosomiasis control professionals with snail survey experiences of 6 years and less (75%) and more than 6 years (90%) (χ2 = 7.792, P < 0.05). The accuracy, sensitivity, specificity and AUC of the AI-enabled intelligent snail identification system were 88%, 100%, 76% and 0.88 for recognition of O. hupensis robertsoni snail sample images, and there was no significant difference in the accuracy of recognition of O. hupensis robertsoni snail sample images between the AI-enabled intelligent snail identification system and artificial identification (χ2 = 0.204, P > 0.05). In addition, there was no significant difference in the accuracy of artificial identification for recognition of snail sample images with upward (90%) and downward shell openings (86%) (χ2 = 0.379, P > 0.05), and there was a significant difference in the accuracy of artificial identification for recognition of snail sample images between schistosomiasis control professionals with snail survey experiences of 6 years and less and more than 6 years (χ2 = 5.604, Padjusted < 0.025). Conclusions The accuracy of recognition of snail sample images is comparable between the AI-enabled intelligent snail identification system and artificial identification by schistosomiasis control professionals, and the AI-enabled intelligent snail identification system is feasible for recognition of O. hupensis robertsoni and Tricula in Yunnan Province.
10.Research on The Construction and Application of Multiple Fluorescence Amplification System for Three Kinds of Stains
Yi-Fan BAI ; He-Miao ZHAO ; Jing CHEN ; Hong-Di LIU ; Rui-Qin YANG ; Chong WANG
Progress in Biochemistry and Biophysics 2025;52(4):982-994
ObjectiveA multiplex amplification system was constructed based on the capillary electrophoresis platform for simultaneous detection of saliva, semen, and vaginal secretions using tissue-specific RNA markers. The aim of this study is to identify the tissue origin of suspicious body fluid stains found at crime scenes and determine whether the body fluid stains at the crime scene are one or several types among saliva, semen, and vaginal secretions. MethodsThirty saliva samples, forty semen samples, and forty vaginal secretion samples (half from 2015 and half from 2024) were collected from healthy adult volunteers. Through primer designing, system formulation, and PCR condition optimization, a multiplex fluorescent amplification system was constructed. The specificity, sensitivity, and detection ability for mixed samples of this system were investigated, and it was tested using real crime scene materials. In the primer design stage, to reduce the requirements for RNA template quality, the amplification products were set within 80-300 bp. In the system formulation stage, dominant and subordinate primers were mainly considered. By reducing the concentration of dominant primers and increasing that of subordinate primers, a capillary electrophoresis spectrum with an appropriate peak height ratio was finally obtained. Additionally, gradient experiments were designed to adjust the concentrations of PCR reagents and PCR amplification conditions, and multiple versions of DNA amplification enzymes were optimized to achieve the best experimental results. ResultsThrough statistical analysis, there was no significant difference in the capillary electrophoresis of the 3 types of body fluid samples from the two years (2015 and 2024), demonstrating that the sample preservation method in this study can preserve samples for a relatively long time. The composite amplification system constructed in this study exhibited high specificity for all 3 types of body fluid, with no cross-reactions between the markers of each type of body fluid. The minimum detection thresholds for the 3 types of body fluid reached 0.002 9, 0.001 5, and 0.42 mg/L, respectively. This system also had a high degree of discrimination for mixed samples, especially for semen-saliva mixtures, where each body fluid marker could still be successfully detected when the concentration ratio of semen to saliva was 100:1. Meanwhile, in the two actual cases presented in this article, the application of this composite amplification system performed outstandingly. ConclusionThe composite amplification detection system constructed in this study can achieve the correct screening of saliva, semen, and vaginal secretions, overcoming the problems such as low specificity and sensitivity of marker tests and unbalanced RFU values of each marker in previous studies. The specificity and sensitivity meet the practical work requirements, and the operation is simple. It provides an analytical and identification method for body fluid stains in actual case and is applicable to the identification of the tissue origin of biological evidence at crime scenes involving sexual assault, indecent assault, and other criminal acts. In the future, more types of body fluid markers will be screened to expand the types of body fluids detected by the system, and body fluid-specific cSNP and cInDel genetic markers will be introduced to infer the sources (individuals and types) of mixed and complex stains more accurately.

Result Analysis
Print
Save
E-mail