应用种特异性PCR技术快速鉴定辣椒实蝇

【目的】辣椒实蝇 Bactrocera latifrons (Hendel)为我国重要的检疫性有害生物,其寄主范围广泛,危害严重。由于传统鉴定方法受到饲养周期、饲养条件、虫态等因素的限制,使得果蔬进出口贸易通关速度、疫情快速鉴定受到较大的影响,因此迫切需要开发关于实蝇的快速鉴定识别的技术。【方法】本研究基于mtDNA COI序列设计了一对能够准确鉴定辣椒实蝇的种特异性引物FL680和RL1057,选用辣椒实蝇作为阳性对照,选用番石榴实蝇B. correcta (Bezzi)、桔小实蝇 B. dorsalis (Hendel)和颜带实蝇 B. cilifer (Hendel)等20种实蝇作为阴性对照,进行PCR扩增并将PCR产物进行电泳检测。【结果】仅目标种辣椒实蝇能够扩增出清晰且单一的约378 bp的条带,其余实蝇种类均未出现条带。将本实验建立的种特异性PCR（SS-PCR）鉴定方法应用于实际检疫工作中并得到了验证,表明该方法具有强的种特异性。【结论】本文提出辣椒实蝇快速鉴定识别技术可应用于实蝇的疫情监测和口岸的检疫检测工作。     

Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis

A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10(4) to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.     

Molecular identification of Liriomyza trifolii (Burgess) (Dipt., Agromyzidae) based on real-time PCR

Abstract:  Rapidly identifying juvenile individuals of Liriomyza trifolii (Burgess) from Liriomyza sativae Blanchard is crucial in plant quarantine. We report a molecular method to identify L . trifolii based on real-time polymerase chain reaction (PCR). By comparing partial DNA sequences of mitochondrial COI genes of L . trifolii samples collected from Guangdong and Taiwan provinces in China, Japan, Philippine, Israel, Germany, the USA, Mexico and Honduras sequenced by authors, and those of related species recorded in GenBank, a L . trifolii -specific probe was developed. There was no difference in individuals of different stages tested by this probe. The total time for real-time PCR assay system was 2 h, and it would save 3–7 h compared with conventional PCR.     

Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution

We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses.     

Detection of the solanum fruit fly, Bactrocera latifrons (Hendel) in Tanzania (Dipt., Tephritidae)

Abstract:  The presence of the Solanum fruit fly, Bactrocera latifrons , in Africa is reported for the first time, based on trapped and reared specimens in Tanzania. Two new host records, Solanum aethiopicum and Solanum macrocarpon , are reported.     

Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples

Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.     

橘小实蝇快速检疫鉴定方法

采用形态学观察与PCR技术相结合的方法,针对台湾水果中截获的可疑橘小实蝇Bactroceradorsalis(Hendel)幼虫和蛹进行快速鉴定。采用异硫氰酸胍方法快速提取昆虫基因组DNA,并根据特异性引物,采用PCR技术从截获的可疑橘小实蝇幼虫和蛹样本中均扩增得到224 bp的特异性条带,经测序比较发现,扩增产物序列与橘小实蝇目的基因序列完全相同,由此证明可疑样本为橘小实蝇的幼虫和蛹。该方法解决了橘小实蝇幼虫和蛹等未成熟虫态的快速检疫难题,大大缩短了检测周期,值得口岸检验检疫借鉴应用。     

SYBR Green I荧光RT-PCR法检测贝类中的诺如病毒

针对诺如病毒II型的保守区域设计引物,建立了SYBR Green I实时荧光RT-PCR检测诺如病毒II型的反应体系。此方法的病毒检测下限达到102拷贝,标准曲线的线形范围为102~106拷贝,相关系数为0.9952,斜率为?2.982,截距为35.84。对诺如病毒II型检测特异,与轮状病毒、腺病毒、甲肝病毒、星状病毒无交叉反应。针对质粒标准品检测的批内试验变异系数 (CV) 为0.95％~1.69％ (n=5),批间试验CV为0.87％~1.24％ (n=3)。运用此方法随机检测30份贝类水产品,检测出2份阳性样品。结果表明,SYBR Green I荧光RT-PCR检测诺如病毒II型的方法灵敏、特异、重复性好,可应用于贝类水产品的快速检测。     

SYBR green dye-based probe-free SNP genotyping: Introduction of T-Plex real-time PCR assay

Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T_m discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.     

SYBR Green实时荧光定量PCR快速检测口腔幽门螺杆菌

目的建立一种快速、灵敏、特异的鉴定幽门螺杆菌实时荧光定量PCR方法。方法利用SYBR Green实时荧光定量PCR反应体系对口腔幽门螺杆菌进行检测。鉴定结果与临床常规鉴定方法相比较,评价其敏感度、特异度及重复性。结果通过48例样品的检测,结果显示实时荧光定量PCR法检测标本的鉴定结果与常规PCR鉴定方法的结果对比,特异度为100%,敏感度为100%;最小能检测到102个拷贝数的重组质粒;批内重复试验和批间重复试验结果均与常规鉴定方法结果相符。结论实时荧光定量PCR法鉴定口腔幽门螺杆菌,特异度和敏感度高,重复性好,且快速、简便。该方法有望成为检测口腔幽门螺杆菌感染的一种快速有效的方法。     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

实时荧光定量PCR快速鉴别新型冠状病毒主要变异株北大核心CSCD

为建立一种快速鉴别严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的5种主要变异株的Taq Man探针实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)体系,基于SARS-CoV-2野生型及变异株alpha (N501Y、HV69-70del)、beta (E484K、K417N)、gamma (K417T、V1176F)、delta (L452R、T478K)和omicron (H655Y、N679K、P681H)序列设计特异性引物、探针,建立和优化一种鉴别新型冠状病毒(SARS-CoV-2) 5种主要变异株的Taq Man探针RT-qPCR方法,并进行该方法的特异性、敏感性、鉴别能力评价。该方法可准确区分出SARS-CoV-2野生型和突变型,与其他呼吸道病原体(n=21)无交叉,显示高特异性。该方法最低检测限为2×10;拷贝/mL,操作简单、快速、成本廉价,可用于监测SARS-CoV-2毒株的变异,精准指导疫情识别与防控。     

基于mtDNA Cytb 的六种果实蝇的分子鉴定

本研究首次对果实蝇属的桔小实蝇Bactrocera dorsalis、瓜实蝇B. cucurbitae、南瓜实蝇B. tau、番石榴实蝇B. correcta、具条实蝇B. scutellata、黑漆实蝇B. scutellaris等6种实蝇mtDNA Cytb基因进行了测序。对这6种实蝇72个个体mtDNA Cytb基因中段420 bp的碱基序列进行分析,得到38种单倍型,发现了116个变异位点,其中30个位点较为稳定。对这6种实蝇与其各自鉴别位点的对应关系研究表明,mtDNA Cytb基因可以作为这6种实蝇种类鉴别的分子标记。     

Alcohol dehydrogenase allele frequencies in Bactrocera oleae (Dipt., Tephritidae): effect of ethanol addition in larval diet

Abstract: The effect of ethanol in larval medium on Bactrocera oleae larvae was examined at four concentrations. Ethanol exerted a differential effect on the three alcohol dehydrogenase allele frequencies. While originally being at equilibrium under laboratory conditions, after three generations of larval development in a diet containing ethanol at 1% concentration, Adh -F allele frequency increased, that of Adh -I dropped significantly and the frequency of Adh -S remained unaltered. Adh -S allele seems to be adapted in nature where only minor quantities of alcohol are present in the insects' natural host, while Adh -I is best adapted in the alcohol-free laboratory culture medium. The frequency of Adh -F allele remains unaltered when feral populations are introduced in the laboratory.     

柑橘大实蝇内参基因的评估

【目的】柑橘大实蝇Bactrocera minax (Enderlein)是一种危害严重的柑橘害虫。本研究旨在筛选柑橘大实蝇在特定条件下体内稳定表达的内参基因, 以确保使用实时荧光定量PCR分析目标基因表达的可靠性。【方法】选择10种候选内参基因用于进行实时荧光定量PCR（qRT-PCR）, 利用5种软件对柑橘大实蝇在不同虫态下（低龄幼虫、3龄幼虫、1日龄蛹、80日龄蛹、160日龄蛹、雄成虫、雌成虫）以及成虫不同部位（成虫头、胸、腹、整体）中候选内参基因的Ct值进行分析, 明确其表达的稳定性。【结果】在柑橘大实蝇不同虫态和成虫不同部位, 10种候选内参基因的Ct值都处于15~30之间, 各基因Ct值的不同表明各基因的表达量存在差异。 综合分析各种软件对内参基因稳定性的排名, 结合geNorm软件对最佳内参基因数量的分析结果, 推荐在不同虫态下采用UBQ, GAPDH和GST作为内参基因, 在不同成虫部位中采用TUB, GAPDH和GST作为内参基因。【结论】为了获取可信的目标基因表达分析结果, 建议根据不同条件选择使用不同的内参基因组合。本研究结果有利于进一步研究柑橘大实蝇在特定条件下的目标基因表达。     

SYBR green实时荧光定量PCR检测微小RNA21的方法建立及在食管鳞癌中的应用

目的:建立SYBR green实时荧光定量PCR检测微小RNA miR-21的技术平台及应用。方法:设计微小RNA21和U6的的颈环结构反转录引物和PCR扩增引物,以U6为内参利用SYBR green实时荧光定量PCR法检测小鼠各器官中的微小RNA21的含量。提取16例食管鳞癌患者的肿瘤组织及其近旁组织中的总RNA,检测其微小RNA21表达水平。结果:SYBR green实时荧光定量PCR检测U6和微小RNA21含量的熔解曲线单一,PCR产物特异。在Balb/c小鼠的4种器官中,肝脏、脾脏、肾脏分别为脑组织的8.71、5.38、3.47倍。16对食管鳞癌患者的样本中,14例微小RNA21的拷贝数高于其近旁组织约10.58倍(p0.01)。结论:此研究成功建立了SYBR green荧光定量PCR法检测小鼠和人微小RNA-21含量的技术平台,为进一步阐述miR-21在食管鳞癌的发生中的作用提供了新方向。     

桔小实蝇肌球蛋白轻链2基因的克隆及表达分析

肌球蛋白轻链在生物的运动过程中具有重要作用, 本研究利用RT-PCR和RACE技术获得了桔小实蝇Bactrocera dorsalis （Hendel）肌球蛋白轻链2 (myosin light chain 2, MLC2)的cDNA全长序列, 命名为BdorMLC2。测序结果表明, BdorMLC2开放阅读框全长669 bp, 编码222个氨基酸残基。在线软件SMART分析显示, BdorMLC2具有2个Ca²⁺结合基序（EFh）结构域, 可以结合Ca²⁺, 属于肌钙蛋白C超家族成员。氨基酸序列比对表明, BdorMLC2与其他昆虫的肌球蛋白具有较高的序列一致性, 其中与黑腹果蝇Drosophila melanogaster MLC2的序列一致性高达93.2%。BdorMLC2在不同组织和时期的荧光定量PCR分析表明, BdorMLC2在桔小实蝇雄虫胸部的含量最高, 在前足、中足、后足和翅中也有较高的表达; BdorMLC2几乎在桔小实蝇发育的各个时期都有表达, 刚羽化成虫的表达量显著高于其他发育阶段。结果提示, BdorMLC2可能与桔小实蝇的肌肉收缩运动密切相关。本研究为深入研究桔小实蝇肌球蛋白的功能提供了理论依据。     

桔小实蝇超氧化物歧化酶基因SOD3的克隆及表达分析

采用反转录聚合酶链式反应(RT-PCR)和快速扩增c DNA末端(RACE)技术克隆桔小实蝇SOD3基因,并命名为Bdor SOD3。Bdor SOD3阅读框全长531 bp,编码176个氨基酸,第1-20位氨基酸为其信号肽区域;该蛋白序列与桔小实蝇的另外一种SOD蛋白AGE89778.1序列的一致性最高,达98.7%;采用Swiss-model在线软件模拟构建Bdor SOD3蛋白的三维结构;采用半定量PCR方法,研究Bdor SOD3基因大肠杆菌诱导后的表达情况,结果表明,Bdor SOD3在处理与对照的24 h、48 h都有表达,但Bdor SOD3在处理后48 h表达量明显升高,结果暗示Bdor SOD3与桔小实蝇蛹对大肠杆菌的免疫机制有关。     

Seasonality and host utilization of the invasive fruit fly, Bactrocera invadens (Dipt., Tephritidae) in central Tanzania

Abstract:  The temporal occurrence of the invasive and economically important pest fruit fly, Bactrocera invadens was studied in three agro-ecological areas of Morogoro Region, central Tanzania, during 2004–2005. Weekly and monthly trappings were carried out with methyl eugenol, protein bait and synthetic food attractant. Bactrocera invadens was permanently present at low and mid-altitudes (380–520 m a.s.l.) with peak periods coinciding with the fruiting season of mango ( Mangifera indica ) and guava ( Psidium guajava ). At high altitude (1650 m a.s.l.) its incidence was only temporal and apparently the result of dispersal from lower altitudes after the mango fruiting season. Rearing results showed mango, loquat ( Eriobotrya japonica ), guava and grapefruit ( Citrus  ×  paradisi ) to be the favoured commercial host fruits. Other Citrus species, cucurbits, papaya ( Carica papaya ) and avocado ( Persea americana ) were less favoured.