Cardiovascular health awareness,risk perception,behavioural intention and INTERHEART risk stratification among middle-aged adults in Malaysia

Objective:To investigate the interrelationship between cardiovascular health awareness,risk perception,behavioural intention,and INTERHEART risk stratification in a middle-aged adult population in Malaysia.Methods:A cross-sectional survey with convenience sampling was conducted during November 2022 and January 2023.Participants completed validated questionnaires assessing cardiovascular health awareness,risk perception of cardiovascular diseases,behavioural intention towards adopting healthy habits,and INTERHEART risk stratification score(IHRS)based on established risk factors.A total of 602 respondents were included in the final analysis.Data were analysed with independent t-test/one-way ANOVA or Mann-Whitney/Kruskal-Wallis to test the differences,Pearson correlation or linear regression test to analyze the association of independent and dependent variables.Results:There was a significant positive correlation between medical knowledge related to cardiovascular disease(CVD)and knowledge related to CVD risk prevention,risk perception,behavioural intention and IHRS(P<0.05,Pearson correlation).Notably,individuals with higher IHRS tended to have lower knowledge related to CVD and CVD risk prevention,risk perception,and behavioural intention.Males,laborers,active/former smokers,individuals with lower household income and educational levels,those involved in occupations not related to the healthcare sector,and those who did not receive the CVD health brochure or are unaware of health self-assessment tools are likely to have lower levels of knowledge,risk perception,and poorer behavioural intention regarding cardiovascular health(P<0.05,one-way ANOVA).While educational level,smoking status,awareness about CVD poster,self-assessment tools were repeatedly significantly associated with knowledge related to CVD and CVD risk prevention,risk perception,behavioral intention and/or IHRS(P<0.05,linear regression).Conclusions:These findings underscore the importance of promoting cardiovascular health awareness and risk perception among middle-aged adults to foster positive BI and reduce CVD risk.Tailored interventions targeting specific risk factors identified by INTERHEART may enhance risk stratification accuracy and facilitate targeted preventive strategies.

Correlation between psychological traits and the use of smart medical services in young and middle-aged adults: An observational study

BACKGROUND Psychological problems affect economic development.However,there is a huge gap between mental health service resources and mental health service needs.Existing mental health service technology and platforms cannot meet all the diverse mental health needs of people.Smart medicine is a new medical system based online that can effectively improve the quality and efficiency of medical services and make mental health services accessible.AIM To explore the level of intelligent medical use among young and middle-aged people and its correlation with psychological factors.METHODS Convenience sampling was used to select 200 young and middle-aged patients with medical experience at the Third People's Hospital of Chengdu between January 2022 and January 2023 as the research subjects.The general condition Questionnaire,Eysenck Personality Questionnaire,Symptom Checklist-90,General Health Questionnaire,and Smart Medical Service Use Intention Questionnaire were used to collect data.Pearson’s correlation was used to analyze the correlation between the participants’willingness to use smart medical services and their personality characteristics,psychological symptoms,and mental health.RESULTS The results revealed that the mental health of young and middle-aged people was poor,and some had psycho-logical problems such as anxiety,depression,and physical discomfort.Familiarity,acceptance,and usage of smart healthcare in this population are at a medium level,and these levels correlate with psychological characteristics.Acceptance was positively correlated with E,and negatively correlated with P,anxiety,fear,anxiety/insomnia,and social dysfunction.The degree of use was negatively correlated with P,obsessive-compulsive symptoms,depression,anxiety,hostility,paranoia,and somatic symptoms.CONCLUSION The familiarity,acceptance,and usage of smart medical services among the middle-aged and young groups are related to various psychological characteristics.

Study on the association of multimorbidity with sleep and diet in young and middle-aged adults in Chongqing region,China

Background:The prevalence of multimorbidity is increasing,with a notable shift towards younger age groups.Methods:Convenience sampling was employed to select 15,988 young and middle-aged adults in Chongqing as the study population.The Apriori algorithm was used to identify the multimorbidity patterns within this population,while an unordered binary logistic regression model was applied to assess the association between sleep,diet and the risk of multimorbidity.Results:The study population had an average age of 39.2±10.1 years.Apriori association rules revealed that the most prevalent binary chronic disease multimorbidity pattern was dyslipidaemia+hypertension(14.78%),while the most common ternary chronic disease multimorbidity pattern was diabetes+dyslipidaemia+hypertension(9.66%).The logistic regression analysis revealed that a lower rating of sleep quality was associated with an elevated risk of multimorbidity(odds ratio(OR)=1.17,95%confidence interval(CI):1.01,1.36).Individuals who adhere to a meat-based diet(OR=1.45,95%CI:1.25,1.68)and those with elevated salt intake(OR=1.22,95%CI:1.01,1.47)were also observed to have an increased risk of multimorbidity.Additionally,a greater likelihood of multimorbidity was observed among those following a spicy diet(OR=1.3,95%CI:1.11,1.52)and consuming more oil(OR=1.16,95%CI:1.01,1.33).Conclusion:A poor sleep quality and a dietary preference for meat-based,salt,spicy and oils were found to be associated with an increased risk of multimorbidity progression among young and middle-aged populations.It is recommended that young and middle-aged adults should pay attention not only to sleep duration but also to sleep quality and improve dietary habits to reduce the likelihood of multimorbidity.

Reading man皮瓣修复术治疗皮肤缺损的疗效观察

目的:探讨Reading man皮瓣修复术在全身不同部位皮肤缺损及其预防瘢痕增生的临床疗效。方法:回顾性分析2021年1月—2023年10月该科应用Reading man皮瓣修复术治疗全身不同部位皮肤软组织缺损的20例患者临床资料。结果:所有患者皮瓣均成活。术后随访时间为3~24个月,所有皮瓣血运良好,色泽红润,质地柔软,外观适宜,局部皮肤张力较低,手术切口瘢痕增生不明显,切口周围软组织无明显形变,周围关节活动度正常。所有患者均恢复良好,疗效满意。结论:Reading man皮瓣特有的设计可以很好地解决全身多部位较大的皮肤软组织缺损,尤其在术中可以最大限度地减少正常皮肤的切除,术后外观、功能及皮肤瘢痕增生均有较满意的表现,且不存在自体皮移植带来的供皮区损伤。

牛冠状病毒TaqMan荧光定量PCR检测方法的建立及应用

为建立牛冠状病毒TaqMan荧光定量PCR检测方法,根据GenBank收录的牛冠状病毒AKS-01株N基因序列(KU886219)保守区设计特异性引物和探针,构建重组质粒并进行反应条件优化、特异性试验、重复性试验以及敏感性试验,建立一种检测BCoV的TaqMan荧光定量PCR方法。结果显示,建立的牛冠状病毒TaqMan荧光定量PCR检测方法特异性、敏感性和重复性均良好。该方法BCoV重组质粒标准品在5.75×10^(7)~5.75×10^(3)copies/μL时与Ct值呈现良好线性关系,该方法对牛轮状病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒、牛副流感病毒3型均无交叉反应,特异性良好;该方法对BCoV重组质粒标准品最低检测限为5.75×10^(1)copies/μL;批内和批间重复性试验结果稳定,变异系数均小于2%。利用所建立的TaqMan荧光定量PCR方法对收集的132份样品进行检测,与常规PCR相比,两者符合率为96.21%,可为BCoV的临床检测和流行病学调查提供技术支持。

大口黑鲈弹状病毒Taq Man荧光定量PCR检测方法的建立及初步应用

为建立快速、敏感的大口黑鲈弹状病毒(MSRV)流行株Taq Man荧光定量PCR (qPCR)方法,本研究采用NCBI分析MSRV流行株N基因的保守序列,设计特异性引物和Taq Man探针,以构建的并经PCR和测序鉴定正确的重组质粒标准品p MD18-MSRV-N为模板,通过优化各反应条件初步建立MSRV的Taq Man q PCR检测方法。结果显示,建立的Taq Man q PCR方法标准曲线的R2为0.9906,质粒标准品在6.79×10^(8)拷贝/μL~6.79×10^(2)拷贝/μL范围内与Ct值均存在较好的线性关系。采用该方法检测MSRV及相关病毒,评估该方法的特异性,结果显示,该方法可以特异性检测MSRV,而草鱼出血病病毒、神经坏死病毒、鲤春病毒血症病毒、大口黑鲈双RNA病毒、鳜蛙虹彩病毒、传染性脾肾坏死病毒、石斑鱼虹彩病毒、大口黑鲈蛙虹彩病毒等8种常见鱼类病毒检测结果均为阴性;采用建立的方法检测不同浓度的质粒标准品(6.79×10^(8)拷贝/μL~6.79×10^(1)拷贝/μL),评估该方法的敏感性,结果显示,该方法的检测限为6.79×10^(2)拷贝/μL,比文献中的常规PCR方法敏感1 000倍;以不同批次或同一批次提取的不同浓度的质粒标准品作为模板,分别利用该方法检测,评估该方法的重复性,结果显示,该方法批内和批间重复性试验的变异系数均小于2%。采用该方法和文献中的常规PCR同时检测71份临床发病的大口黑鲈、鳜鱼、鲮鱼组织样品,结果显示阳性率为16.9%(12/71),阴性率为83.1%(59/71),且阳性样品均是从发病大口黑鲈的组织样品中检测到,而发病的鳜鱼、鲮鱼组织样品中均未检测到该病毒;常规PCR方法的阳性率为9.9%(7/71),阴性率为90.1%(64/71),二者的总符合率为93.0%。本研究建立的MSRV Taq Man q PCR方法特异性强、敏感性高、重复性和准确性均较好,可用于大口黑鲈临床样品的检测,为MSRV的快速检测提供了可行的技术手段。

The Relationship between Mental Health Literacy and Subjective Well-Being of Young and Middle-Aged Residents: Perceived the Mediating Role of Social Support and Its Urban-Rural Differences

This study investigates mental health literacy among young and middle-aged urban and rural residents and the differences in mental health literacy,perceived social support and subjective well-being.From January to February 2022,620 participants(320 rural and 300 urban residents)from three provinces of China were selected by con-venience sampling.A general data questionnaire,mental health literacy scale,perceived social support scale and subjective well-being scale were administered.The mental health literacy scores of urban residents were 3.34±0.57 and those of rural residents were 2.73±0.79.The results of multiple regression analysis showed that the mental health literacy scores of rural residents were more significant than those of urban residents in terms of sex and age,while urban residents’mental health literacy scores were more significant than those of rural resi-dents in terms of monthly income.Mental health literacy,perceived social support and subjective well-being of young and middle-aged urban and rural residents were all positively correlated(P<0.01).While the level of mental health literacy is better among urban residents than rural residents,perceived social support plays a partial mediating role between mental health literacy and subjective well-being of both urban and rural residents,and should be the focus of researchers seeking to improve the level of well-being of residents.

鸡毒支原体 Taq Man实时荧光定量PCR检测方法的建立

为快速检测鸡毒支原体,根据鸡毒支原体的保守基因设计特异性引物和Taq Man探针,建立鸡毒支原体Taq Man实时荧光定量PCR方法并分析鸡毒支原体培养过程中颜色单位改变法(color change unit,CCU)和荧光定量PCR检测的相关性。结果显示,标准曲线y=-3.646x+44.208,相关系数R^(2)=0.998,最低检测限度为1 copy/μL;与其他菌株等均无交叉反应;组内和组间变异系数均小于3%,表明该方法具有良好的稳定性和可重复性。用建立的实时荧光定量PCR检测方法对26份临床样品进行检测,该方法较普通PCR方法检出率更高。建立的荧光定量PCR与CCU显示鸡毒支原体在对数生长期时,两者具有一定的对应关系。表明建立了鸡毒支原体Taq Man探针实时荧光定量PCR检测方法,可实现对鸡毒支原体的快速检测,为鸡毒支原体感染的防控提供技术基础。

牛支原体和丝状支原体丝状亚种双重TaqMan荧光定量PCR检测方法的建立与应用

牛传染性胸膜肺炎和牛支原体病均为重要的牛传染病,两者均可致牛出现呼吸系统症状,临床上难以区分。为建立鉴别诊断牛传染性胸膜肺炎和牛支原体病的双重Taq Man荧光定量PCR检测方法,本研究根据这两种病的病原丝状支原体丝状亚种和牛支原体的基因组保守区域(丝状支原体丝状亚种nt826-nt1742,牛支原体nt189-nt618)分别设计引物与探针,通过优化反应体系与反应条件,建立同时检测这两种病原的Taq Man荧光定量PCR检测方法。特异性试验结果显示,该方法仅对丝状支原体丝状亚种模拟样品和牛支原体检测结果为阳性,而巴氏杆菌、牛传染性鼻气管炎病毒、无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、关节炎支原体Leachii株检测结果均为阴性,特异性较强。分别以1.0×10~7拷贝/μL~1.0×10~1拷贝/μL的p EASY-Mmm和p EASY-Mb质粒标准品为模板,进行敏感性试验,结果显示,该方法对丝状支原体丝状亚种和牛支原体重组质粒标准品的检测限均为1.0×10~1拷贝/μL,敏感性较高。对不同浓度质粒标准品混合物的重复性试验结果显示,组内与组间重复性试验变异系数均小于2.5%,重复性较好。利用该方法对112份临床样品(104份鼻拭子、8份肺组织样品)检测,结果显示,丝状支原体丝状亚种检测结果和已发表PCR方法检测结果一致,均为阴性,而本实验建立的方法检测出4份牛支原体阳性样品,且经测序证实检出样品为真实阳性样品,而已发表的多重PCR方法检测该病原结果均为阴性。本研究建立了能够同时检测丝状支原体丝状亚种和牛支原体的双重Taq Man荧光定量PCR方法,其特异性强、敏感性高、重复性好,可用于各种临床样品的检测,为这两种病原的快速检测和流行病学调查提供了技术手段。

羊贝氏柯克斯体TaqMan荧光定量PCR检测方法的建立及应用

为建立检测贝氏柯克斯体(Coxiella burnetii)实时荧光定量PCR方法,根据GenBank上收录的C.burnetii RSA439株com1基因序列(CP040059.1)保守区设计特异性引物和探针,并构建重组质粒及优化反应条件,以期建立一种检测贝氏柯克斯体的Taq Man荧光定量PCR方法。结果显示,该方法所建立标准曲线线性关系良好;敏感性试验结果显示,最低检出限为2.45×10^(2) copies/μL,与PCR最低检出限为2.45×10^(4) copies/μL相比较,灵敏度提高了100倍;对羊支原体、羊流产衣原体、羊布鲁氏菌等均无交叉反应,批内和批间重复性试验结果稳定,变异系数均小于3%,说明特异性、重复性好;利用该方法对收集的126份样品进行检测,结果显示样品阳性率为9.52%,常规PCR检测阳性率为7.14%,符合率为90.47%。该方法可为贝氏柯克斯体临床上快速检测及防控提供技术支持。

Case of clear-cell hepatocellular carcinoma that developed in the normal liver of a middle-aged woman

A 36-year-old woman was admitted to our department for close examination of a liver tumor that was found during a medical checkup. Abdominal US, CT and MRI showed a tumor in segment 7 (S7) of the liver. Although imaging suggested hepatocellular carcinoma, laboratory tests showed no abnormality in liver function, hepatitis virus markers were negative, and tumor markers including protein induced by vitamin K absence or antagonist Ⅱ (PIVKA-Ⅱ), α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA) were all within normal ranges. Upon aspiration biopsy of the liver, the histopathological diagnosis was moderately differentiated hepatocellular carcinoma. Therefore, right hepatectomy was performed. Although a part of the tumor was necrotic, about 60% of the viable part showed a clear-cell variant. Consequently, it was diagnosed as clear-cell hepatocellular carcinoma. It was noted that the background liver tissue was normal. This case is worthy of reporting because development of clear-cell hepatocellular carcinoma in the normal liver of a middle-aged woman is rarely seen.

Effect of Age and Sex on Stroke Mortality of Young and Middle-aged Adults in China,2002–2019,and Predictions to 2030

Objective This study aimed to examine the trends in stroke mortality among young and middle-aged adults in China.Methods Data were obtained from the China national vital registration system.Significant changes in mortality were assessed by Joinpoint regression.Age-period-cohort analysis was used to explain the reasons for the changes.Future mortality and counts were predicted by the Bayesian age-period-cohort model.Results Between 2002 and 2019,a total of 6,253,951 stroke mortality in young and middle-aged adults were recorded.The age-adjusted mortality rates(AAMRs)of women showed a downward trend.The annual percent changes(APC)were-3.5%(-5.2%,-1.7%)for urban women and-2.8%(-3.7%,-1.9%)for rural women.By contrast,the AAMRs per 100,000 for rural men aged 25–44 years continued to rise from 9.40 to 15.46.The AAMRS for urban men aged 25–44 years and urban and rural men aged 45–64years did not change significantly.Between 2020 and 2030,the projected stroke deaths are 1,423,584 in men and 401,712 in women.Conclusion Significant sex and age disparities in the trends of stroke mortality among young and middle-aged adults were identified in China.Targeted health policy measures are needed to address the burden of stroke in the young generation,especially for rural men,with a focus on the prevention and management of high risk factors.

猪肺炎支原体和猪鼻支原体TaqMan双重荧光PCR检测方法的建立及应用

为建立一种可快捷鉴别检测猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)的方法,针对Mhp的183基因和Mhr的p37基因保守片段,分别设计合成引物及TaqMan探针,优化反应条件,建立一种可同时检测Mhp和Mhr的TaqMan双重荧光PCR方法,并评价其特异性、敏感性和重复性。结果表明,该方法特异性强,检测其他常见猪呼吸系统病原不发生交叉反应;对标准品pMD18-T-Mhp-183、pMD18-T-Mhr-P37的最低检测限分别达45.2 copies/μL和29.7 copies/μL,比普通PCR检测灵敏度提高10^(3)~10^(4)倍;该方法检测结果的批内与批间变异系数均小于2%。用该方法检测145份广西自治区内临床样品,从中检出82份Mhp和9份Mhr阳性样品。该方法可用于临床样品快速检测及实验室Mhp和Mhr的鉴定,可为Mhp和Mhr在猪群中的早期监测提供技术支持。

Relationship between Waist Circumference, Waist-Height Ratio, Body Fat Percentage, Visceral Fat and Bone Mineral Density in Middle-Aged and Elderly Zhuang of China

Objective: To understand the correlation between different degrees of obesity and bone mineral density (BMD). Methods: 772 cases (300 men and 472 females) of Zhuang over 40 years old were randomly selected. The BMD and T score were calculated by measuring the right heel bone using a quantitative-ultrasound densitometer. Body composition index of body weight, waist circumference (WC), waist height ratio (WtHR), body fat percentage (BFP) and visceral fat (VF) were measured using a bioelectrical impedance method. Results: The BMI, WC, and WtHR of the male osteoporosis group were lower than those of the normal bone mass (NBM) group (P Conclusion: BMI is positively correlated with BMD in Zhuang. BFP has a greater correlation with female BMD, and a small reduction in BFP can result in a significant decrease in BMD. BMD of male was positively correlated with abdominal obesity indicators (WC, WtHR, VF). However, abdominal obesity has no obvious effect on the promotion of BMD in women, and increased WtHR can lead to a decrease in BMD. Zhuang can appropriately increase the amount of fat within the normal weight range. At the same time, reasonable exercise and balanced nutrition to avoid excessive obesity or low body weight can reduce the incidence of osteoporosis and osteoporosis fractures.

circMAN1A2调节miR-212-3p/DDIT4轴对胃癌细胞增殖、侵袭和迁移的影响

目的探究circMAN1A2靶向miR-212-3p/DNA损伤诱导转录子4(NDA damage-inducible transcript 4,DDIT4)轴对胃癌细胞增殖、侵袭和迁移的影响。方法qRT-PCR法检测胃癌组织和细胞中circMAN1A2、miR-212-3p表达水平。荧光素酶检测circMAN1A2与miR-212-3p、DDIT4和miR-212-3p的靶向关系。在胃癌细胞SGC-7901、HGC-27中转染si-NC、si-circMAN1A2、si-circMAN1A2+anti-NC、si-circMAN1A2+anti-miR-212-3p、miR-NC、miR-212-3p mimics,将未转染的SGC-7901、HGC-27细胞设为对照。平板克隆实验检测细胞增殖;细胞划痕及Transwell实验分别检测细胞迁移、侵袭能力。Western blotting检测增殖、迁移及侵袭相关蛋白表达。结果在胃癌组织及细胞中,circMAN1A2高表达,miR-212-3p低表达;下调circMAN1A2可以靶向上调miR-212-3p表达,且下调DDIT4表达,进而抑制胃癌细胞增殖、侵袭和迁移能力。抑制miR-212-3p表达可部分逆转抑制circMAN1A2表达对胃癌细胞恶性增殖的抑制作用。结论在胃癌细胞中抑制circMAN1A2表达可抑制胃癌细胞增殖、迁移与侵袭,其与调节miR-212-3p/DDIT4信号轴有关。

猪细小病毒TaqMan荧光定量PCR检测方法的建立及应用

旨在建立一种猪细小病毒(PPV)快速准确PCR检测方法,根据PPV VP2基因的保守序列设计并合成1对特异性引物和1条特异性探针,建立了可检测PPV的TaqMan荧光定量PCR检测方法。建立的检测方法敏感性高,最低可检测8.76×10^(1)copies/μL的PPV VP2基因标准品;特异性试验结果显示,该方法检测猪流行性腹泻病毒、猪圆环病毒2型、伪狂犬病病毒、猪繁殖与呼吸综合征病毒均无交叉反应;重复性试验组内与组间变异系数均低于2%,重复性良好。用该方法检测102份临床样本,检出阳性样本16份,阳性检出率为15.69%,将检测出的阳性样本进行PCR扩增并测序,证实检测样品中确实存在PPV。该方法敏感性高、特异性强,适用于PPV的定量检测及猪细小病毒病的流行病学调查。

Association of fatigue with short-term prognosis in young and middle-aged patients with coronary heart disease

Background:To investigate the effect of fatigue on the short-term prognosis of young and middle-aged patients with coronary heart disease(CHD).Methods:A cross-sectional,observational survey was distributed at a tertiary hospital in Suzhou,China.Patients were assessed for fatigue and their prognosis was assessed at 3 and 6 months after discharge.General Information Questionnaire,Fatigue Scale and Seattle Angina Questionnaire(SAQ)were used for the survey.The Cox proportional hazard model was used to analyze the impact of fatigue on the occurrence of major adverse cardiac events(MACEs)at 3 and 6 months after discharge.Multiple linear regression models were used to analyze the effect of fatigue on health-related quality of life(HRQoL)at 3 and 6 months after discharge.Results:199 patients were followed up with in the end.43 patients(21.6%)with MACE three months after discharge had a total SAQ score of(399.76±39.61).The overall SAQ score was(425.14±22.66)and 52 patients(26.1%)experienced MACE six months after discharge.Fatigue was identified as a risk factor for MACE 6 months after discharge by the Cox proportional hazard model(HR=2.939,95%CI:0.177~0.655,P=0.001)and as an independent risk factor for quality of life 3 and 6 months after discharge by multiple linear regression(P<0.001).Conclusions:In individuals who are young or middle-aged and have coronary heart disease,fatigue is a risk factor for the short-term prognosis.It is advised that clinical professionals prioritize patient fatigue assessment and improve management of fatigue symptoms.

Influencing factors of fatigue in young and middle-aged patients with coronary heart disease:a cross-sectional study

Objective:To examine the present state of fatigue in young and middle-aged patients with coronary heart disease(CHD)and to identify the elements that may be affecting it.Methods:From December 2020 to June 2021,240 young and middle-aged patients with CHD who were being treated in the cardiology department of a tertiary care hospital in Suzhou were chosen using a convenience sampling method in order to gather data on the patients’sociodemographic status,fatigue,social support,and stress.Results:Of the 240 disseminated questionnaires,220 valid responses were returned,resulting in an effective recovery rate of 91.67%.The frequency of exhaustion was 51.8%,and the fatigue score was(5.27±2.77).The stress score was(11.15±3.36),while the overall social support score was(39.13±4.72).Binary logistic regression analysis indicated that age,exercise,staying up late,stress,social support,high-sensitivity troponin T,high-density lipoprotein,and ejection fraction were independent risk factors for fatigue in young and middle-aged patients with CHD(P<0.05).Conclusions:Fatigue is more prevalent in young and middle-aged patients with CHD.Clinical nurses can create a unique management plan for patients based on their lifestyle and behavioral patterns,stress levels,social support,and clinical signs to reduce fatigue.

虾十足目虹彩病毒1、肝肠胞虫及传染性早熟病毒多重Taq Man荧光定量PCR检测方法的建立与应用

为建立检测十足目虹彩病毒1(DIV1)、肝肠胞虫(EHP)、传染性早熟病毒(IPV)的多重Taq Man荧光定量PCR检测方法,本研究根据DIV1 MCP基因、EHP SSU基因与IPV polyprotein A基因设计特异性引物和Taq Man探针,经各反应条件的优化建立了同时检测DIV1、EHP及IPV的多重Taq Man荧光定量PCR方法。利用该方法检测DIV1、EHP、IPV、白斑综合征病毒、致急性肝胰腺坏死弧菌、传染性皮下及造血器官坏死病毒、虾偷死病毒、传染性肌肉坏死病毒、桃拉病毒、黄头病毒1型等主要虾类病毒,结果显示,该方法仅能扩增出DIV1、EHP和IPV,与其他主要虾类病原均无交叉反应,特异性较强。对10倍倍比稀释后等浓度等体积混合的DIV1、EHP和IPV质粒标准品混合物的检测结果显示,该方法对3种病原的质粒标准品的检测限均为1.67×10^(1)拷贝/μL,各病毒单一荧光定量PCR方法的检测限均为1.67×10~0拷贝/μL,多重Taq Man荧光定量PCR方法的灵敏度虽略低于单一荧光定量PCR方法,但是可以同时检测3种病原,检测效率高。选取3个浓度的pDIV1、pEHP、pIPV(5×10^(5)拷贝/μL、5×10^(4)拷贝/μL、5×10^(3)拷贝/μL)等浓度等体积混合的质粒标准品为模板,进行组内和组间重复性试验,结果显示组内与组间重复性试验的变异系数均小于1%,该方法重复性较好。利用该方法与DIV1、EHP、IPV单一荧光定量PCR方法同时检测105份虾苗和亲虾样品,结果显示,本研究建立的多重Taq Man荧光定量PCR方法对DIV1的检出率为21.9%(23/105),对EHP的检出率为14.3%(15/105),对IPV的检出率为45.7%(48/105),3种病原混合感染率为1.9%(2/105),与DIV1、EHP、IPV各单一荧光定量PCR方法的检测结果均一致,阳性样品的符合率达100%。本研究建立的DIV1、EHP、IPV多重Taq Man荧光定量PCR方法为临床同时检测该3种病原提供了特异、敏感、高效的技术手段。

TaqMan探针双重实时荧光定量PCR法同步检测兰花中的建兰花叶病毒和齿兰环斑病毒

为建立一种同步定量检测建兰花叶病毒(cymbidium mosaic virus,CymMV)和齿兰环斑病毒(odontoglossum ringspot virus,ORSV)的高效检测方法,本研究根据CymMV和ORSV CP基因高度保守区分别设计引物和探针并筛选,获得156 bp和148 bp的靶标序列及对应的最优特异性引物探针组合,建立了基于TaqMan探针的双重实时荧光定量PCR方法。该方法最低检出限为1拷贝或6.2×10^(-3)fg,最低稳定检出限为10拷贝或6.2×10^(-2)fg,是RT-PCR的10~100倍;构建的标准曲线线性关系良好,扩增效率分别为97.7%和100.2%,相关系数R2分别为1.000和0.999;对其他5种常见病毒均无扩增曲线,检测特异性强;批组内与批组间重复性试验Ct值变异系数≤0.60%,重复性和稳定性好。利用该方法和基因芯片法分别对4个种属的66个兰花样品进行方法验证,该方法相较于基因芯片法检出率提高了53.85%(CymMV)和162.5%(ORSV)。综上所述,本方法可同时高通量检测CymMV和ORSV 2种病毒靶基因,结果可靠,应用前景广阔,可为开展病毒精准鉴定、科学防控以及从源头遏制病毒传播提供技术支撑。