1.Analysis of Coagulation Changes and Influencing Factors during Treatment of Acute Promyelocytic Leukemia.
Zhen-Zhu CHEN ; Tao LIU ; He-He GUO ; Wen-Wen REN ; Kai WANG ; Ying-Xu PANG
Journal of Experimental Hematology 2025;33(1):45-53
OBJECTIVE:
To analyze the changes in coagulation during the treatment of acute promyelocytic leukemia (APL) and explore the influencing factors of coagulation in patients with APL.
METHODS:
Data of 166 APL patients admitted to our hospital from November 2018 to May 2023 were retrospectively analyzed, and the changes of various clinical indicators before and during treatment were compared. 166 APL patients were divided into abnormal coagulation group (n =115) and normal coagulation group (n =51) according to whether they experienced coagulation dysfunction. The basic information, clinical data and laboratory indicators of the two groups were compared. Multivariate logistic regression analysis was used to screen risk factors for coagulation dysfunction and established logistic regression model. Then we developed a neural network model and ranked the importance of the influencing factors, and used receiver operating characteristic (ROC) curves to evaluate the predictive performance of the two models.
RESULTS:
The comparative results of various clinical indicators in 166 APL patients before and during treatment showed that systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-C), estimated glomerular filtration rate (eGFR), platelet (PLT) and fibrinogen (FIB) were significantly increased during the treatment (P < 0.05), while glycosylated hemoglobin (HbA1c), high density lipoprotein cholesterol (HDL-C), blood urea nitrogen (BUN), serum creatinine (SCr), high-sensitivity C reactive protein (hs-CRP), IL-6, TNF-α, TGF-β, white blood cells (WBC), absolute neutrophil count (ANC), prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer (D-D), fibrinogen degradation products (FDP) and lactate dehydrogenase (LDH) were significantly decreased during the treatment (P < 0.05). The proportion of patients with hemorrhage and high-risk APL in the abnormal coagulation group was significantly higher than that in the normal coagulation group (P < 0.05). The levels of IL-6, TNF-α, WBC, ANC, D-D, FDP and LDH in the abnormal coagulation group were significantly higher than those in the normal coagulation group (P < 0.05). The influencing factors selected by univariate analysis were incorporated into logistic regression analysis and neural network model to predict the risk of coagulation dysfunction in APL patients. ROC curves showed that the AUC of the two models were 096 and 0.908, the sensitivity were 0.824 and 0.892, the specificity were 0.940 and 0.904, the Youden index were 064 and 0.796, and the accuracy were 0.882 and 0.898, respectively.
CONCLUSION
High risk stratification, hemorrhage, elevated WBC, LDH, ANC and FDP levels are independent risk factors for coagulation dysfunction in APL patients. The logistic regression model and neural network model based on these risk factors demonstrate good predictive performance for coagulation dysfunction in APL patients.
Humans
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Leukemia, Promyelocytic, Acute/therapy*
;
Blood Coagulation
;
Retrospective Studies
;
Male
;
Female
;
Risk Factors
;
Logistic Models
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Middle Aged
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Adult
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ROC Curve
2.Development and evaluation of a triplex RT-qPCR assay with internal references for detection of the Dengue and Zika viruses
Meng-Tao CAO ; Xiao-Yu HU ; Wei YANG ; Chun-Yuan LI ; Xiao-Li XU ; Rui-Wen REN ; Hong-Xia JIANG
Chinese Journal of Zoonoses 2024;40(6):537-543
A triplex RT-qPCR assay with human genes as internal references was established for detection of the Dengue and Zika viruses(DENV and ZIKV,respectively).The conserved regions of the four serotypes of DENV,along with the NS1 gene of ZIKV and the human β-actin gene,which is stably ex-pressed in various human tissues,were targeted by three sets of specific primers and probes.Standard plasmids for four se-rotypes of DENV,ZIKV,and β-actin were constructed as pos-itive controls.Optimal reaction conditions were determined through an L9(34)orthogonal experiment.The specificity,sensitivity,and coverage of the assay were verified and evalua-ted clinically,and the consistency was evaluated against a com-mercial kit for detection of DENV.The triplex RT-qPCR assay established exhibited no non-specific cross reactions with 12 similar arboviruses.The detection sensitivity for DENV and ZIKV were 2.99 and 2.18 copies/μL,respectively,and the intra-group and inter-group repeatability coefficients of variation were within 1.5%.As compared to the commercial kit,the proposed assay obtained positive results for 13 epidemic strains of DENV.Bland-Altman consistency analysis confirmed that the consistency of the detection results of clinical positive samples between the commercial kit and the proposed assay was 92.59%.The highly specific and sensitive triplex RT-qPCR assay with internal references is an effective tool for early and rapid differential identification of DENV and ZIKV.
3.Effects of Different Test Positions on Quantitative Muscle Strength of Wrist and Finger Flexor Muscle Groups and Its Standardization
Ze-Ren YANG ; Dong GAO ; Qing XIA ; Dan RAN ; Yan-Liang SHENG ; Wen-Tao XIA
Journal of Forensic Medicine 2024;40(3):237-244,253
Objective To explore the effects of different test positions on quantitative muscle strength of wrist and finger flexor muscle groups and to establish a standardized muscle strength test protocol for each muscle group.Methods Forty healthy subjects (12 males and 28 females) were recruited.A portable digital quantitative muscle strength tester,Micro FET2TM,was used to measure the flexor muscle strength of each finger and the wrist joint at the 30° extension,0° neutral,and 30° flexion,respectively.Palmar abduction strength of the thumb was measured at 30° and 60°,respectively.Ten subjects were randomly selected from the 40 subjects,and the quantitative muscle strength of each muscle group was tested again by the same operator after an interval of 10 to 15 days.Results Except for the fact that in males,there was no significant difference in flexor muscle strength of thumb and wrist joint between 30° of wrist extension and neutral 0° position,the muscle strength of the other fingers flexion and wrist palmar flexor showed the following characteristics:30° of wrist extension>neutral 0° posi-tion>30° of flexion,and the PAST was 30°>60°;The flexor muscle strength of all the subjects was thumb>index finger>middle finger>ring finger>little finger;All muscle strength values of male were greater than those of female,and the difference was statistically significant (P<0.05);There was no significant difference between the left and right side muscle strength values of all subjects (P>0.05).The reliability of muscle strength values measured at different times in 10 subjects was good.Conclu-sion The quantitative muscle strength of each muscle group of the hand and wrist is affected by the test position,and a standardized and uniformed test position should be adopted in the actual identification.Micro FET2TM has good reliability for hand and wrist quantitative muscle strength testing.The 30° ex-tension of the wrist can be used as the best standardized test position for the flexion muscle strength of each finger and wrist joint.The 30° position can be used as the best standardized test position for PAST.
4.Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients (version 2024)
Yao LU ; Yang LI ; Leiying ZHANG ; Hao TANG ; Huidan JING ; Yaoli WANG ; Xiangzhi JIA ; Li BA ; Maohong BIAN ; Dan CAI ; Hui CAI ; Xiaohong CAI ; Zhanshan ZHA ; Bingyu CHEN ; Daqing CHEN ; Feng CHEN ; Guoan CHEN ; Haiming CHEN ; Jing CHEN ; Min CHEN ; Qing CHEN ; Shu CHEN ; Xi CHEN ; Jinfeng CHENG ; Xiaoling CHU ; Hongwang CUI ; Xin CUI ; Zhen DA ; Ying DAI ; Surong DENG ; Weiqun DONG ; Weimin FAN ; Ke FENG ; Danhui FU ; Yongshui FU ; Qi FU ; Xuemei FU ; Jia GAN ; Xinyu GAN ; Wei GAO ; Huaizheng GONG ; Rong GUI ; Geng GUO ; Ning HAN ; Yiwen HAO ; Wubing HE ; Qiang HONG ; Ruiqin HOU ; Wei HOU ; Jie HU ; Peiyang HU ; Xi HU ; Xiaoyu HU ; Guangbin HUANG ; Jie HUANG ; Xiangyan HUANG ; Yuanshuai HUANG ; Shouyong HUN ; Xuebing JIANG ; Ping JIN ; Dong LAI ; Aiping LE ; Hongmei LI ; Bijuan LI ; Cuiying LI ; Daihong LI ; Haihong LI ; He LI ; Hui LI ; Jianping LI ; Ning LI ; Xiying LI ; Xiangmin LI ; Xiaofei LI ; Xiaojuan LI ; Zhiqiang LI ; Zhongjun LI ; Zunyan LI ; Huaqin LIANG ; Xiaohua LIANG ; Dongfa LIAO ; Qun LIAO ; Yan LIAO ; Jiajin LIN ; Chunxia LIU ; Fenghua LIU ; Peixian LIU ; Tiemei LIU ; Xiaoxin LIU ; Zhiwei LIU ; Zhongdi LIU ; Hua LU ; Jianfeng LUAN ; Jianjun LUO ; Qun LUO ; Dingfeng LYU ; Qi LYU ; Xianping LYU ; Aijun MA ; Liqiang MA ; Shuxuan MA ; Xainjun MA ; Xiaogang MA ; Xiaoli MA ; Guoqing MAO ; Shijie MU ; Shaolin NIE ; Shujuan OUYANG ; Xilin OUYANG ; Chunqiu PAN ; Jian PAN ; Xiaohua PAN ; Lei PENG ; Tao PENG ; Baohua QIAN ; Shu QIAO ; Li QIN ; Ying REN ; Zhaoqi REN ; Ruiming RONG ; Changshan SU ; Mingwei SUN ; Wenwu SUN ; Zhenwei SUN ; Haiping TANG ; Xiaofeng TANG ; Changjiu TANG ; Cuihua TAO ; Zhibin TIAN ; Juan WANG ; Baoyan WANG ; Chunyan WANG ; Gefei WANG ; Haiyan WANG ; Hongjie WANG ; Peng WANG ; Pengli WANG ; Qiushi WANG ; Xiaoning WANG ; Xinhua WANG ; Xuefeng WANG ; Yong WANG ; Yongjun WANG ; Yuanjie WANG ; Zhihua WANG ; Shaojun WEI ; Yaming WEI ; Jianbo WEN ; Jun WEN ; Jiang WU ; Jufeng WU ; Aijun XIA ; Fei XIA ; Rong XIA ; Jue XIE ; Yanchao XING ; Yan XIONG ; Feng XU ; Yongzhu XU ; Yongan XU ; Yonghe YAN ; Beizhan YAN ; Jiang YANG ; Jiangcun YANG ; Jun YANG ; Xinwen YANG ; Yongyi YANG ; Chunyan YAO ; Mingliang YE ; Changlin YIN ; Ming YIN ; Wen YIN ; Lianling YU ; Shuhong YU ; Zebo YU ; Yigang YU ; Anyong YU ; Hong YUAN ; Yi YUAN ; Chan ZHANG ; Jinjun ZHANG ; Jun ZHANG ; Kai ZHANG ; Leibing ZHANG ; Quan ZHANG ; Rongjiang ZHANG ; Sanming ZHANG ; Shengji ZHANG ; Shuo ZHANG ; Wei ZHANG ; Weidong ZHANG ; Xi ZHANG ; Xingwen ZHANG ; Guixi ZHANG ; Xiaojun ZHANG ; Guoqing ZHAO ; Jianpeng ZHAO ; Shuming ZHAO ; Beibei ZHENG ; Shangen ZHENG ; Huayou ZHOU ; Jicheng ZHOU ; Lihong ZHOU ; Mou ZHOU ; Xiaoyu ZHOU ; Xuelian ZHOU ; Yuan ZHOU ; Zheng ZHOU ; Zuhuang ZHOU ; Haiyan ZHU ; Peiyuan ZHU ; Changju ZHU ; Lili ZHU ; Zhengguo WANG ; Jianxin JIANG ; Deqing WANG ; Jiongcai LAN ; Quanli WANG ; Yang YU ; Lianyang ZHANG ; Aiqing WEN
Chinese Journal of Trauma 2024;40(10):865-881
Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.
5.Effectiveness of 13-valent pneumococcal conjugate vaccine against invasive disease caused by serotype 19A in children: a meta-analysis.
Zhao Jun LU ; Yan LIU ; Jian DU ; Jun WANG ; Xin Ren CHE ; Wei JIANG ; Xiao Ping ZHANG ; Wen Wen GU ; Yu Yang XU ; Xue Chao ZHANG ; Jing WANG ; Qi Xin XIE ; Ying Ying YANG ; Lin Tao GU
Chinese Journal of Preventive Medicine 2023;57(12):2181-2187
Objective: Using Meta-analysis to evaluate the vaccine effectiveness of 13-valent pneumococcal conjugate vaccine (PCV13) against invasive Streptococcus pneumoniae disease (IPD) caused by serotype 19A in children <5 years old. Methods: "Streptococcus pneumoniae infection""invasive pneumococcal disease""13-valent pneumococcal polysaccharide conjugate vaccine""PCV13""effectiveness""infant""child" and related terms were searched from China National Knowledge Infrastructure (CNKI), WANFANG DATA, PubMed, SCOPUS and Web of science with no limited on language, region and research institution. The retrieval time was limited from January 2010 to February 2023 and cohort study, case-control study and randomized controlled trial were included. Data were extracted from eligible studies by two independent reviewers, and after study quality assessment by NOS scale, Meta-analysis was completed using Stata 16.0 software. Results: A total of 2 340 related literatures were searched, and 10 literatures were finally included, including 5 case-control studies and 5 indirect cohort studies, which showed good literature quality. The vaccine effectiveness against serotype 19A IPD of PCV13 in children was 83.91% (95%CI: 78.92%-88.89%), and the subgroup analysis (P=0.240) showed there was no significant difference among the case-control study (VE=87.34%, 95%CI:79.74%-94.94%) and the indirect cohort study (VE=81.30%, 95%CI:74.69%-87.92%). The funnel plot and Egger test suggested that the possibility of publication bias was small. Conclusion: The present evidence indicates that PCV13 has a good vaccine effectiveness against serotype 19A IPD in children, and it is recommended to further increase the vaccination rate of PCV13 to reduce the disease burden of IPD in children <5 years old.
Child
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Humans
;
Child, Preschool
;
Case-Control Studies
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Cohort Studies
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Serogroup
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Vaccines, Conjugate/therapeutic use*
;
China
;
Pneumococcal Infections/prevention & control*
6.Effectiveness of 13-valent pneumococcal conjugate vaccine against invasive disease caused by serotype 19A in children: a meta-analysis.
Zhao Jun LU ; Yan LIU ; Jian DU ; Jun WANG ; Xin Ren CHE ; Wei JIANG ; Xiao Ping ZHANG ; Wen Wen GU ; Yu Yang XU ; Xue Chao ZHANG ; Jing WANG ; Qi Xin XIE ; Ying Ying YANG ; Lin Tao GU
Chinese Journal of Preventive Medicine 2023;57(12):2181-2187
Objective: Using Meta-analysis to evaluate the vaccine effectiveness of 13-valent pneumococcal conjugate vaccine (PCV13) against invasive Streptococcus pneumoniae disease (IPD) caused by serotype 19A in children <5 years old. Methods: "Streptococcus pneumoniae infection""invasive pneumococcal disease""13-valent pneumococcal polysaccharide conjugate vaccine""PCV13""effectiveness""infant""child" and related terms were searched from China National Knowledge Infrastructure (CNKI), WANFANG DATA, PubMed, SCOPUS and Web of science with no limited on language, region and research institution. The retrieval time was limited from January 2010 to February 2023 and cohort study, case-control study and randomized controlled trial were included. Data were extracted from eligible studies by two independent reviewers, and after study quality assessment by NOS scale, Meta-analysis was completed using Stata 16.0 software. Results: A total of 2 340 related literatures were searched, and 10 literatures were finally included, including 5 case-control studies and 5 indirect cohort studies, which showed good literature quality. The vaccine effectiveness against serotype 19A IPD of PCV13 in children was 83.91% (95%CI: 78.92%-88.89%), and the subgroup analysis (P=0.240) showed there was no significant difference among the case-control study (VE=87.34%, 95%CI:79.74%-94.94%) and the indirect cohort study (VE=81.30%, 95%CI:74.69%-87.92%). The funnel plot and Egger test suggested that the possibility of publication bias was small. Conclusion: The present evidence indicates that PCV13 has a good vaccine effectiveness against serotype 19A IPD in children, and it is recommended to further increase the vaccination rate of PCV13 to reduce the disease burden of IPD in children <5 years old.
Child
;
Humans
;
Child, Preschool
;
Case-Control Studies
;
Cohort Studies
;
Serogroup
;
Vaccines, Conjugate/therapeutic use*
;
China
;
Pneumococcal Infections/prevention & control*
7.Experimental study on the structural and functional changes of the retinal tissue induced by intense blue light irradiation in rats
Wen-Jing WANG ; Shuai SHENG ; Jian-Tao REN ; Xu-Dong HUANG
International Eye Science 2023;23(11):1787-1792
AIM: To observe the structural and functional changes of retinal tissue in rats after different duration of intense blue light irradiation.METHODS: A total of 48 healthy 8-week-old SD male rats were selected and randomly divided into the control group(n=12)and 3, 6 and 12h experimental groups(n=36). The rats in the control group received natural light, and the rats in the experimental groups received blue light with a wavelength of 465±5nm and the illumination of 1000±100lx for 3, 6, and 12h each day. Optical coherence tomography(OCT), fundus fluorescein angiography(FFA)and haematoxylin-eosin(HE)staining of paraffin pathological section were used to observe the changes of the retinal thickness, retinal tissue structure and the function in different directions and layers.RESULTS: The OCT results showed that the retinal thickness in the superior, inferior, nasal, and temporal sides of rats in different groups was statistically significant(P<0.05), and there was no statistical significant difference between the control group and the 3h experimental group in the total retinal thickness(P>0.05), while the differences between any other two groups were statistically significant(P<0.05); The mean total retinal thickness, internal limiting membrane(ILM)-inner nuclear layer(INL)thickness, outer plexiform layer(OPL)-outer segment(OS)thickness and retinal pigment epithelium(RPE)of rats in each group were statistically significant(P<0.05), and the mean total retinal thickness and OPL-OS thickness were statistically significant between any two groups(P<0.05). The ILM-INL thickness of rats in the control group and 3 and 12h experimental groups was statistically significant(P<0.05), and the thickness of RPE layer in the 12h experimental group was statistically different from that of the 3 and 6h experimental groups(P<0.05). FFA results showed that there was no obvious fluorescence leakage in the fundus of rats in the control group and the 3h experimental group, while there was obvious fluorescence leakage and hyperfluorescence in the retina of the 6 and 12h experimental groups, and the background fluorescence of choroid was enhanced. HE staining showed the atrophy and apoptosis of cells in the optic cell layer, and some lightly stained nucleus. In addition, RPE layer showed atrophy and thinned with the increase of light time, and there was significant difference in the number of the optic cells between the control group and experimental group(P<0.05).CONCLUSION: The intense blue light irradiation could cause thinning of the retina in rats, with varying degrees of thinning in different layers of the retina. It could also led to decrease and even disappearance of the number of cells in the visual cell layer, the focal atrophy of the RPE layer, and the change of vascular permeability. With the extension of the light time, the structural and functional changes in retinal tissue became more obvious.
9.Optimization of production process of Aeromonas veronii CA07 strain inactivated vaccine
SUN Cheng⁃wen ; GONG Hua ; LAI Ying⁃tiao ; JIANG Xiao⁃yan ; REN Yan ; CHEN Zong⁃hui ; HUANG Zhi⁃bin ; TAO Jia⁃fa
Chinese Journal of Biologicals 2023;36(1):76-80+84
Abstract:Objective To optimize the production process of inactivated vaccine of Aeromonas veronii(AV)CA07 strain.
Methods The fermentation culture process of AV CA07 strain liquid was determined through the optimization of the culture
time(2~16 h),medium(optimized fermentation medium,LB medium and NB medium)and fermentation conditions(in⁃
oculation amount of 1%,5%,10% and 15%;ventilation rate of 2,4,6 and 8 L/min and fermentation time of 6,8,10
and 12 h). The optimal inactivation process was determined through the comparison of the final concentration of formalde⁃
hyde solution(0. 10%,0. 20%,0. 30% and 0. 40%),inactivation temperature(28 and 37 ℃)and inactivation time(24,
48 and 72 h). The large⁃scale production process of inactivated vaccine of AV CA07 strain in 500 L fermentor was estab⁃
lished and the prepared vaccines were tested for safety and immunogenicity. Results The optimal inoculation amount of AV
CA07 strain was 5%,ventilation rate was 4 L/min and culture time was 10 ~ 12 h. The optimal inactivation condition was
adding formaldehyde solution with final concentration of 0. 30% incubating at 37 ℃ for 24 h. The number of viable bacteria
in the fermentation broth of AV CA07 strain prepared in 500 L fermentor was more than 8 × 109 CFU/mL. All crucian carps
immunized with the inactivated vaccine by abdomen survived. After challenge,the relative immune protection rate was more
than 90%. Conclusion AV CA07 strain inactivated vaccine prepared by optimized production process showed good safety
and immunogenicity.
10. Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus
LI Chun-yuan ; LIU Jiong ; LIU Ji-ru ; HU Xiao-yu ; GAO Meng-tao ; CHEN Yue ; TIAN Jing ; REN Rui-wen ; XU Xiao-li
China Tropical Medicine 2023;23(2):121-
Abstract: Objective To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.


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