Rapid Preparation of Filamentous Fungal DNA for PCR Analysis by Ultrasonic Treatment

A simple method to prepare of DNA template suitable for PCR amplification from filamentous fungi will be valuable for improving experimental efficiency.Here,a method was developed which just needed ultrasonic treatment of the mycelium at usual condition,and the produced solution could directly be used as DNA template for internal transcribed spacer(ITS)amplification successfully.The PCR could be improved by additional treatment of 60℃water baths,but was not centrifugation.When the template amount was 0.5-2μL and the ultrasonic time was 7-11 min,there was no distinctly influences on PCR.The method was commonly used for M.purpureus,I.cicadae,Lentinula sp.,Flammul sp.and Dictyophora sp.etc.to detect target sequences of ITS,hygromycin resistance gene(Hyg),CRISPR-associated protein 9(Cas9),Citrinin gene C(CtnC),Citrinin gene D(CtnD),large subunit rRNA gene(NL),and so on.The method could provide a simple,rapid,safe and economic approach to prepare the DNA template for large-scale PCR of the special filamentous fungi materials.

Study on PCR rapid molecular detection technique of Meloidogyne vitis

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS(ribosomal DNA internal transcribed spacer)gene fragment as the target.The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established.The result showed that the optimal annealing temperature of the primers was 53℃,which was suitable for the detection of different life stages of M.vitis.Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile(J_(2))and 10^(-4)female.Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples.The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J_(2)of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J_(2)s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green co ntrol strategies.

微滴式数字PCR技术快速诊断侵袭性念珠菌病的方法建立

目的建立基于微滴式数字PCR(droplet digital PCR,ddPCR)技术快速检测重症患者侵袭性念珠菌感染的方法。方法基于微滴式数字PCR平台建立检测体系,设计四种念珠菌(白色念珠菌、光滑念珠菌、近平滑念珠菌和热带滑念珠菌)特异性引物探针;(1)对无模板对照(no tmplate contral,NTC)样品进行检测,确定空白限(limit of blank,LOB)范围和阳性判断值;(2)通过对阳性样本进行稀释,每个浓度梯度各进行10次重复提取检测,确定检测限(limit of detection,LOD)范围;(3)重复检测稀释后的样本,确定定量限(limit of quantitation,LOQ)范围;(4)对阳性样品进行梯度稀释,确定线性范围;(5)通过2个高低浓度的阳性样本提取检测,进行12次重复性测试,计算结果浓度对数值的变异系数(CV);(6)通过具有真菌培养结果的临床样本进行检测,评估方法可靠度。结果ddPCR检测念珠菌LOB在0~81 copies/mL之间,阳性判断值为≥3个阳性微滴;LOD为3×10^(2) copies/mL;LOQ为3×10^(2) copies/mL;不同浓度梯度检测线性范围是3×10^(2)~3×10^(7)copies/mL,相关系数为:白色念珠菌R^(2)=0.9995、光滑念珠菌R^(2)=0.9989、近平滑念珠菌R^(2)=0.9994、热带滑念珠菌R^(2)=0.999;结果浓度对数值的CV<5%,满足精密度要求;通过检测建立方法初步验证临床标本,结果与临床培养结果一致。结论ddPCR对重症患者侵袭性念珠菌感染的检测灵敏度高、重复性好,特异性高。

Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture(Most probably Number)

Objective:To analyse molecular detection of coliforms and shorten the time of PCR.Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number(MPN)method for 16 artificial and 101 field samples.The molecular method was also conducted on isolated coliforms from positive MPN samples;standard sample for verification of microbial method certificated reference material;isolated strains from certificated reference material and standard bacteria.The PCR and electrophoresis parameters were changed for reducing the operation time.Results:Results of PCR for lacZ and uidA genes were similar in all of standard,operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR.PCR results were confirmed by MPN culture method by sensitivity 86%(95%CI:0.71-0.93).Also the total execution time,with a successful change of factors,was reduced to less than two and a half hour.Conclusions:Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city.It's recommended to be used at least as an initial screening test,and then the positive samples could be randomly tested by MPN.

韦塞尔斯布朗病病毒RT-PCR快速检测方法的建立及应用

为实现对韦塞尔斯布朗病病毒(Wesselsbron virus,WSLV)的快速检测,根据WSLV NS5基因保守序列,设计特异性扩增引物和探针,建立WSLV的TaqMan荧光定量RT-PCR和RT-PCR检测方法,并验证方法的敏感性、特异性及临床可行性。结果显示:所建立的两种方法的核酸检测下限分别为10、100 copies/μL,对日本脑炎病毒、西尼罗病毒和坦布苏病毒核酸均无交叉反应;分别利用建立的两种检测方法对100份入境马匹样品进行检测,未检出WSLV阳性,与南非参考实验室提供的双重荧光RT-PCR检测方法结果一致。结果说明,所建的两种方法敏感性高、特异性强,可用于临床样本检测,为韦塞尔斯布朗病的出入境检验检疫、流行病学调查及防控提供了技术支撑。

水产品中副溶血弧菌、溶藻弧菌和霍乱弧菌三重PCR检测方法的建立

为实现水产食品中多种弧菌同步快速检测,拟建立一种同时检测副溶血弧菌、溶藻弧菌和霍乱弧菌的三重PCR快速检测方法。根据副溶血弧菌的bla CARB-17基因、溶藻弧菌的gyrB基因和霍乱弧菌的toxR基因设计特异性引物。针对3种弧菌分别建立单重PCR体系并优化各项主要反应参数,确定最佳退火温度。然后建立多重PCR体系并优化其反应参数,分析三重PCR的特异性和灵敏度;并验证其实效性。结果表明:三重PCR的最佳退火温度为60℃,能同时扩增出副溶血弧菌bla CARB-17基因的230 bp片段、溶藻弧菌gyrB基因的337 bp片段,霍乱弧菌toxR基因的130 bp片段,灵敏度分别为0.583 ng/μL、0.394 ng/μL、0.866 ng/μL。该三重PCR检测方法可以从实际样品中快速特异地检出三种弧菌,具有较好的特异性,较高的灵敏度,为实际生产中检测水产食品中的弧菌污染提供参考。

实时荧光定量PCR检测猴痘病毒的方法研究

目的建立猴痘病毒(mpox virus)的实时荧光定量PCR(RT-PCR)检测方法。方法以猴痘病毒F3L基因为靶序列,设计特异性引物和探针,建立猴痘病毒RT-PCR检测方法,并对方法的灵敏度、特异性和重复性进行检测;对疑似猴痘阳性的人脓拭子临床样本核酸进行检测,对RT-PCR方法检测临床样本能力进行评价。结果本研究建立的RT-PCR检测方法可实现猴痘病毒的特异性检测,与天花、痘苗、牛痘等其他正痘病毒无交叉反应,检测重复性好,最低检测限为103copies/μL。可以对人皮肤拭子样本中的猴痘病毒进行准确检测。结论本研究建立了猴痘病毒的RT-PCR检测方法,可在临床样本中快速、特异、灵敏的检测猴痘病毒。

Rapid Identification of Methicillin Resistant <i>Staphylococcus aureus</i>Using Real Time PCR

Screening for colonization with methicillin resistant Staphylococcus aureas (MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories using conventional methods is time consuming. In this research we are trying to evaluate the use of real time PCR for the detection of MRSA. The PCR assay was evaluated in clinical isolates of MRSA (n = 45) and methicillin susceptible Staphylococcus aureas MSSA (n = 10). The diagnostic values of the assay showed high sensitivity and specificity. This real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory. Real-time PCR now is available in all laboratories so its use in identification of MRSA will help in shortening the period for MRSA identification and will help in the success of infection control programs in hospitals.

SARS-COV-2 Rapid Antigen Test in Comparison with RT-PCR for Laboratory Diagnosis of COVID-19 in a Southwest State of Nigeria

Objectives: Rapid and accurate identification of persons infected with SARS-CoV-2 which causes COVID-19 is key to managing the pandemic. The urgent need to scale up access to COVID-19 testing in Nigeria has led to the government’s introduction of the use of COVID-19 Ag rapid diagnostic test (RDT) across various settings in the country. However, field performance evaluation of the rapid SARS-CoV-2 antigen detection test is required to be conducted periodically and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. Design: A prospective COVID-19 screening and un-blinded verification of the performance of the STANDARD Q COVID-19 Ag test kit. Setting: The rapid SARS-CoV-2 antigen detection test, StandardTM Q COVID-19 Ag kit was compared with the RT-PCR test for detection of SARS-CoV-2 in nasopharyngeal samples for COVID-19 screening from persons and personnel attending a national youth camp orientation exercise during the second wave of the COVID-19 outbreak (January to March 2021) in Ondo state, southwest Nigeria. Participants: Three hundred fifty-one persons and personnel were screened for COVID-19 infection. Results: Of 351 respondents screened, 68 (19.4%) were positive, and 264 (75.2%) were negative for both COVID-19 Ag RDT and RT-PCR assay. The rapid SARS-CoV-2 antigen detection test’s sensitivity and specificity were 78.16% (95% CI = 68.02% - 86.31%) and 100.0% (95% CI = 98.61% - 100.0%), respectively and the diagnostic accuracy was 94.59% (95% CI: 92 - 97). Respondents that were symptomatic had a higher test sensitivity of 78.6% (49.2 - 95.3) compared to those without symptoms 78.1% (66.9 - 86.9) (p Conclusions: Our study shows evidence that StandardTM Q COVID-19 Ag kit can be an appropriate rapid antigen test that could be used to screen for positive COVID-19 tests to guide decision-making for clinical management of persons infected with COVID-19, especially for closed settings and other clinical care settings.

日勾维多细菌源菌双重PCR快速检测方法建立

建立日勾维多细菌源菌快速检测方法,快速有效地检出样品中的日勾维多细菌源菌。利用日勾维多细菌源菌的看家基因gyrB和ropB,设计2对特异性的扩增引物,以培养液作为DNA扩增模板,建立双重PCR快速检测方法,并通过优化扩增反应的退火温度,提高双重PCR的特异性;利用引物gyrB-F/gyrB-R和ropB-F/ropB-R(insert)扩增4株日勾维多细菌源菌,可得到2条特异性的目标DNA条带,但非目标菌会出现非特异性DNA条带。优化后的退火温度为65℃,以4株日勾维多细菌源菌为DNA模板时,可扩增出特异性的目标DNA条带;以大肠埃希氏菌、金黄色葡萄球菌、铜绿假单胞菌、阴沟肠杆菌和洋葱伯克霍尔德菌为DNA模板时,均检测不到DNA条带。当样品中的日勾维多细菌源菌达到4.4 CFU/mL时,双重PCR即可高灵敏检出样品中的日勾维多细菌源菌。可用双重PCR方法快速检出沐浴露、洗发水等淋洗类化妆品中的日勾维多细菌源菌。

快速PCR方法在食品检验中的应用研究

食品安全关系公众的健康和生命安全,而快速准确的食品检测技术是保障食品安全的重要手段。本文通过阐述快速聚合酶链式反应(Polymerase Chain Reaction,PCR)方法的基本原理,分析其在食源性致病菌、转基因成分、过敏原和真菌毒素检测方面的具体应用。通过利用快速PCR方法,可使检测时间明显缩短,提高了检测灵敏度和特异性,为食品安全检测的发展提供了强有力的技术支撑。

产黄曲霉毒素真菌双重荧光定量PCR检测技术的建立与应用

为实现产黄曲霉毒素真菌的快速检测,本研究建立了检测产黄曲霉毒素真菌的aflR基因和nor-1基因的双重荧光定量PCR方法。分别针对aflR基因和nor-l基因保守序列,各设计2对特异性引物和探针,通过引物对间的有效性实验各筛选出1对最优的aflR引物探针和nor-1引物探针,随后建立并优化双重荧光定量PCR的反应条件,构建标准曲线,测定该方法的重复性、灵敏度和特异性。结果显示,成功筛选出aflR-2和nor1-2 2组引物/探针组合,并优化和确定了引物/探针的最佳反应终浓度。特异性实验结果显示,本研究建立的检测方法仅对产黄曲霉毒素真菌的aflR基因和nor-1基因呈现特异性扩增,对黑曲霉、碳黑曲霉、赭曲霉和镰刀菌等常见产毒真菌的核酸样品均无扩增曲线。建立的方法重复性好,批内和批间变异系数均<3%,双重反应体系的检测灵敏度均低至1×10^(2)copies/μL。利用本研究所建立的方法与ELISA试剂盒分别对80份食品样品进行检测,结果显示,双重荧光定量PCR共检测出9份阳性样品,而ELISA试剂盒只检测出了7份阳性,表明本研究建立的方法具有更高的灵敏度和准确性。

TaqMan实时荧光PCR快速检测与鉴定单核细胞增生李斯特氏菌能力验证样品

利用TaqMan实时荧光PCR技术对单核细胞增生李斯特氏菌能力验证样品进行快速鉴定和验证。依据GB 4789系列标准的第一法对ACAS-PT526能力验证中的样品D15和样品D16进行检测,通过增菌、分离、初筛和鉴定等传统培养法检测。鉴定过程中还使用了鉴定试剂条API Listeria和全自动微生物鉴定系统。同时采用TaqMan实时荧光PCR技术对经过增菌的样品D15与样品D16的培养物和单核细胞增生李斯特氏菌标准菌株的培养物进行快速鉴定。结果表明,经API Listeria试剂条鉴定,样品D15为单核细胞增生李斯特氏菌,鉴定百分率为98.6%,T值为1;经全自动微生物鉴定系统检测,样品D15为单核细胞增生李斯特氏菌,99%可能性;样品D16为金黄色葡萄球菌,99%可能性;经实时荧光PCR鉴定,样品D15 LB1增菌液培养物及李斯特显色培养基平板上的单菌落都具有显著的S型扩增曲线。综上得出,实时荧光PCR与GB 4789.30—2016的第一法试验结果一致,样品D15为单核细胞增生李斯特氏菌检出,样品D16为单核细胞增生李斯特氏菌未检出,TaqMan实时荧光PCR方法可用于微生物的快速检验,尤其是对盲样检测的辅助验证。

一种山茶刺盘孢Colletotrichum camelliae实时荧光定量PCR快速检测方法的建立

【目的】建立一种快速检测茶树炭疽病病原菌山茶刺盘孢(Colletotrichumcamelliae)的实时荧光定量PCR检测方法。【方法】以茶树炭疽病病原菌山茶刺盘孢(C. camelliae)为检测目标,根据刺盘孢属ApMat基因片段设计特异性引物,建立q PCR检测技术体系,验证引物特异性,测试反应的灵敏度,绘制标准曲线,并采集田间病叶测试检测效果。【结果】设计的四组引物对中,引物对CcF/CcR特异性最好,扩增效率最高;用于定量检测C. camelliae的灵敏度为10 pg/μL,DNA浓度的对数(X)与Ct值(Y)之间的线性关系为Y=-3.529 6X+36.938(R^(2)=0.9957,E=92.01);该体系检测C.camelliae溶解曲线对应的特异峰值Tm为(85.5±0.5)℃;用该体系在人工接种C. camelliae的病斑组织和田间病叶中检测到了C. camelliae的存在。【结论】建立的q PCR反应体系可特异性定量检测C. camelliae,并可应用于田间病害的早期诊断。

柔嫩艾美耳球虫和肠致病性大肠杆菌双重PCR检测方法的建立及流行病学调查应用

【目的】建立可同时检测柔嫩艾美耳球虫(Eimeria tenella,E.tenella)和肠致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)的双重PCR检测方法,为临床E.tenella继发EPEC病的检测提供技术支持。【方法】基于E.tenella和EPEC(escV)分别设计2条特异性引物,并通过退火温度、模板浓度和引物浓度等优化多重PCR的反应条件,建立2种病原体的双重PCR检测方法,并运用该方法检测贵州地区490份肉鸡、蛋鸡粪便样品,进行流行病学分析。【结果】该方法所需模板DNA的最低浓度组合为3.5ng/μL,最佳退火温度为62℃,最佳引物浓度组合为0.1 nmol/μL。流行病学调查显示,贵州毕节样本中E.tenella和EPEC的阳性率均为36.36%,混合感染阳性率9.09%;贵州黎平仅有EPEC感染,阳性率为23%;贵州习水样本中未检测出阳性,锦屏样本中E.tenella阳性率为11.11%,EPEC阳性率为22.22%,无混合感染。总样本中E.tenella、EPEC及2种混合感染的阳性率分别为10.20%、18.37%和2.04%。【结论】建立的柔嫩艾美耳球虫(Eimeria tenella,E.tenella)和肠致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)双重PCR检测方法具有特异性强、敏感性高和重复性好的优点。

微滴式数字PCR技术在菜豆晕疫病菌检测中的应用

为了建立菜豆晕疫病菌(Pseudomonas savastanoi pv.phaseolicola)微滴式数字PCR(ddPCR)检测方法,实现菜豆晕疫病菌的高效率、高精度的检疫鉴定和疫情监控,以菜豆晕疫病菌为研究对象,以位点特异性重组酶作为靶标基因,SSRP_F、SSRP_R、SSRP_P为特异性引物与探针,建立菜豆晕疫病菌ddPCR反应体系,结果表明,菜豆晕疫病菌的ddPCR检测体系的绝对定量检测低限为0.6 copies/μL,ddPCR的检测灵敏度比常规PCR高2个数量级。同时研究了种子携带菜豆晕疫病菌是否具有活性,提高了疫情检出率。

PC饮用水桶中粪链球菌快速定性检测实时荧光PCR方法的建立

开发了一种针对PC饮用水桶中粪链球菌快速定性检测的实时荧光PCR方法,该方法基于粪链球菌23S rDNA基因序列,使用特异性引物和探针进行扩增和检测。对其特异性和重复性进行评估,并分别应用该方法和国标方法对PC饮用水桶样品进行了检测。结果表明,该方法能够对PC饮用水桶中粪链球菌进行快速定性检测,可有效缩短检测时间并减少实验操作,具有较高的特异性和重复性。

基于快速PCR方法的人参属药用植物鉴别研究

目的:建立一种准确、快速、高效鉴别人参属药用植物的分子鉴别方法。方法:采集不同产地的人参、西洋参及同属近缘种植物,所有样品进行总DNA的提取并使用mat K片段进行扩增、测序,进行同源比对后根据其变异位点设计西洋参特异性鉴别引物,人参以文献报道的特异鉴别引物为基础,分别采用两步法进行PCR扩增,从而对人参、西洋参及同属近缘种进行鉴别。结果:通过对影响PCR反应时间的退火温度、变性温度、退火时间、变性时间、循环次数等因素进行优化,并对不同型号PCR仪进行考察,分别获得人参、西洋参快速PCR反应程序。在PCR产物中加入SYBR GreenⅠ染料,正品显示出明亮绿色荧光,而近缘种不显示荧光。结论:快速PCR方法可以简单快速鉴别人参、西洋参,为实现药材分子鉴别的现场运用提供技术支撑。

应用PCR快速检测单核细胞增生症李斯特菌的研究

用聚合酶链式反应(PCR)扩增hly基因,检测单核细胞增生症李斯特菌的纯培养物及其模拟污染的生猪肉、水和牛奶。41株单核细胞增生症李斯特菌的PCR结果呈阳性,而其他李斯特菌,包括英诺克李斯特菌、绵羊李斯特菌、西尔李斯特菌、威儿李斯特菌和格氏李斯特菌以及非李斯特菌均未出现特异性片段。这表明所扩增的hly基因3′末段850bp的DNA片段对单核细胞增生症李斯特菌具有较高的特异性。PCR对单核细胞增生症李斯特菌纯培养物的检测低限达10~5cfu·mL^(-1),对16hLEB增菌培养的模拟污染生猪肉、水和牛奶的增菌培养液用PCR捡测,结果检测生猪肉低限达400cfu·kg^(-1)、水和牛奶为400cfu·L^(-1)。整个检测过程只需24h。这表明PCR法对单核细胞增生症李斯特菌的鉴定具有特异性强、灵敏度高、速度快和易操作等优点。

快速PCR技术鉴别中药材蛤蚧的方法研究

目的:建立简单快速鉴别蛤蚧的特异性PCR方法。方法:对比蛤蚧及其伪品细胞色素C氧化酶I亚基基因(CO I)序列,设计蛤蚧特异性PCR鉴别引物,优化特异性PCR条件,对蛤蚧及其7种常见混淆品进行扩增及荧光检测。结果:扩增产物经琼脂糖凝胶电泳和荧光检测,所有蛤蚧药材均能扩增出约400 bp的特异性条带,加入SYBR Green I染料后,在365 nm下出现强烈绿色荧光,混伪品不具特异条带和绿色荧光,鉴别操作可在30 min内完成。结论:快速PCR结合荧光染料检测可快速鉴别蛤蚧及其常见伪品,为蛤蚧的快速鉴定提供了新的思路和方法。