Validation of reference genes for quantitative real‐time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

Drosophila melanogaster is attracted to chemicals produced by fermentation and it is abundantly found in rotten fruits. Considering its habitat, the fruit fly is reported to be tolerant to environmental chemicals. Quantitative real‐time polymerase chain reaction was employed to investigate the expression pattern and physiological function of genes putatively involved in chemical detoxification. In quantitative real‐time polymerase chain reaction assays, normalization of target gene expression with internal reference genes is required. These reference genes should be stably expressed during chemical exposure and in chemical‐free conditions. In this study, therefore, we used two programs (geNorm and BestKeeper) to evaluate the expression stability of five reference genes (nd, rpL18, ef1β, hsp22 and tbp) in female adult flies exposed to various concentrations of methanol and ethyl acetate. Four genes (nd, rpL18, ef1β and tbp) were found to be suitable for use as reference genes in methanol‐treated flies and three genes (ef1β, nd, tbp) were found to be suitable for use as reference genes in ethyl acetate‐treated flies. These results suggested that a combination of two genes among these stably expressed genes can be used for accurate normalization of target gene expression in quantitative real‐time polymerase chain reaction‐based determination of gene expression profiles in D. melanogaster treated with both chemicals.     

Evaluation of the reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the green peach aphid,Myzus persicae

The green peach aphid, Myzus persicae Sulzer (Hemiptera, Aphididae), is an important cosmopolitan pest. Real time qRT‐PCR has been used for target gene expression analysis on M. persicae. Using real time qRT‐PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods (RefFinder, geNorm, Normfinder, BestKeeper and the comparative ΔC_t method). 18s, Actin and ribosomal protein L27 (L27) were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 (L27) was found to be the least stable reference genes for abiotic studies (photoperiod, temperature and insecticide susceptibility). Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT‐PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research.     

苯并噻唑对不同虫态韭菜迟眼蕈蚊的生物活性

【目的】明确室内条件下挥发性化合物苯并噻唑对韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang的生物活性。【方法】采用三角瓶密闭熏蒸法测定了苯并噻唑对韭菜迟眼蕈蚊成虫、 卵、 幼虫和蛹的熏蒸活性; 利用Oxytherm氧电极研究了苯并噻唑对韭菜迟眼蕈蚊成虫呼吸速率的影响; 利用“Y”型嗅觉仪测定了韭菜迟眼蕈蚊成虫对苯并噻唑的行为反应。【结果】苯并噻唑对雌雄成虫处理0.5~2.0 h的LC50分别为0.186~0.052和0.163~0.039 μL/L; 在0.01~0.13 μL/L剂量下对卵熏蒸处理24 h, 第6 天卵孵化率为4.83%~82.39%, 而对照组孵化率为96.97%; 苯并噻唑熏蒸4龄幼虫6~72 h的LC50变化范围为1.247~0.248 μL/L; 0.01~0.09 μL/L剂量熏蒸蛹24 h, 第5天的羽化率为8.17%~69.63%, 对照组羽化率为96.23%。用0.052和0.039 μL/L浓度分别处理雌雄成虫测定2.5 h内呼吸速率的变化, 表现为处理组初始呼吸速率明显高于对照组, 然后逐渐降低至与对照组持平, 最后明显低于相同处理时间对照组呼吸速率。“Y”型嗅觉仪测定结果表明, 苯并噻唑对韭菜迟眼蕈蚊成虫有较强的引诱作用。在0.5 L/min空气流速条件下, 0.5 μL的苯并噻唑对雌雄成虫的引诱率分别为88.33%和78.33%。【结论】苯并噻唑对韭菜迟眼蕈蚊各虫态有很好的毒杀效果, 并对成虫有强烈的引诱作用。     

Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoans

Traditional real-time quantitative polymerase chain reaction protocols cannot be used accurately with symbiotic organisms unless the relative contribution of each symbiotic compartment to the total nucleic acid pool is known. A modified 'universal reference gene' protocol was created for reef-building corals and sea anemones, anthozoans that harbour endosymbiotic dinoflagellates belonging to the genus Symbiodinium. Gene expression values are first normalized to an RNA spike and then to a symbiont molecular proxy that represents the number of Symbiodinium cells extracted and present in the RNA. The latter is quantified using the number of genome copies of heat shock protein-70 (HSP70) amplified in the real-time quantitative polymerase chain reaction. Gene expression values are then normalized to the total concentration of RNA to account for differences in the amount of live tissue extracted among experimental treatments and replicates. The molecular quantification of symbiont cells and effect of increasing symbiont contributions to the nucleic acid pool on gene expression were tested in vivo using differentially infected sea anemones Aiptasia pulchella. This protocol has broad application to researchers who seek to measure gene expression in mixed organism assemblages.     

环境颜色对韭菜迟眼蕈蚊生物学特性的影响

【目的】明确环境颜色对韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang生长发育及繁殖能力等生物学特征的影响。【方法】在室内条件下,利用黑、棕、绿、红和橙5种颜色卡纸模拟自然环境颜色,比较研究了其对韭菜迟眼蕈蚊生长发育和繁殖的影响。【结果】韭菜迟眼蕈蚊发育历期在橙色环境下最长,为26.15 d,黑色环境下最短,为22.35 d;世代存活率在黑色环境下最高,为43.88%,橙色环境下最低,为28.88%;性比在各颜色环境下无显著差异;雌雄成虫寿命均在棕色环境下最长,分别为2.06 d和2.99 d,橙色环境下最短,分别为1.58 d和2.57 d;单雌产卵量在棕色环境下最多,为62.99粒,橙色环境下最少,为45.64粒;种群趋势指数由高到低依次为:黑色(15.90)棕色(15.07)透明色对照(12.93)红色(11.69)绿色(11.67)橙色(8.50)。【结论】环境颜色影响韭菜迟眼蕈蚊生物学特性,黑色及棕色环境有利于其生长发育及繁殖,橙色环境可抑制其生长发育及繁殖。     

Identification of suitable reference genes for quantitative real‐time PCR normalization in blotched snakehead Channa maculata

A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real‐time (qrt‐)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle‐threshold (C_t) values in the qrt‐PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata.     

不同波长LED光源对韭菜迟眼蕈蚊生殖行为的影响

【目的】明确不同波长的LED光源对韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang求偶、交配及繁殖等生殖行为的影响。【方法】采用红(625~630 nm)、橙(600~605 nm)、黄(590~595nm)、绿(525~530 nm)、蓝(455~460 nm)和白(6 000~6 500 k)6种LED光源在韭菜迟眼蕈蚊成虫交配期进行照光处理,观察统计其求偶和交配行为以及单雌产卵量、卵孵化情况和有效后代数量。【结果】韭菜迟眼蕈蚊成虫求偶前期时长在橙光下最长,为28.48 min。求偶率在蓝光下最高,为86%;橙光下最低,为48%。交配期时长在蓝光下最长,为4.59 min;橙光下较短,为4.23 min。单雌产卵量在各波长光源下与对照均无显著差异。卵孵化率在蓝光下最低,仅为43.41%。有效后代数量在蓝光下最低,仅为27.00头;橙光下次之,为43.40头。【结论】LED光源的波长可影响韭菜迟眼蕈蚊的生殖行为,其中橙光(600~605 nm)不利于其求偶、交配和繁殖;蓝光(455~460nm)虽有利于其求偶和交配,但明显抑制其繁殖。     

百合科寄主植物对韭菜迟眼蕈蚊的生物效应

室内分别用百合科韭菜、大葱、大蒜和圆葱饲喂韭菜迟眼蕈蚊Bradysia odoriphaga Yang et Zhang幼虫,研究了4种寄主植物对其生长发育和繁殖的影响;用气-质谱联用分析技术,检测了人工合成大蒜素及大蒜、圆葱和韭菜的乙醇提取物的主要化学成分。结果表明,4种供试寄主植物对韭菜迟眼蕈蚊生长发育和繁殖的影响存在差异,幼虫取食韭菜最有利于其生长发育和繁殖;取食大蒜和圆葱对其生长发育和繁殖表现出明显的不利性,主要表现为幼虫死亡率增加,幼虫期延长,蛹重减轻,单雌产卵量降低;而取食大葱的影响介于取食韭菜与取食大蒜和圆葱之间。大蒜、圆葱的乙醇提取物和人工合成的大蒜素均对韭菜迟眼蕈蚊1龄幼虫有不同程度的杀虫活性,大蒜和圆葱的乙醇提取物（干粉,2 g/mL）稀释100和200倍,处理后48 h对1龄幼虫的校正死亡率分别达54.7%、28.0%和49.4%、22.7%;10%大蒜素稀释500和1 000倍,处理后48 h校正死亡率达100%和80.0%。成分分析表明硫醚类化合物可能是大蒜和圆葱中含有的杀虫活性物质之一。     

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman^® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.     

韭蛆人工饲料配方筛选及饲养效果比较

【目的】研究实验室条件下韭蛆 Bradysia odoriphaga Yang et Zhang大量饲养的方法以解决试虫不足的问题。【方法】本实验在25℃,相对湿度75%,光周期16L:8D条件下,通过控制变量法筛选出人工饲料配方,运用两性生命表软件,分析比较人工饲料与韭菜Allium tuberosum的饲喂效果。【结果】对不同成分和不同含量的8种韭蛆人工饲料配方筛选,结果确定最优的人工饲料配方成分和含量为：平菇粉6 g,韭菜粉5 g,琼脂粉1.25 g,山梨酸0.04 g,苯甲酸0.05 g,酵母粉0.5 g,维生素C 2.5 g和水50 mL。该配方和韭菜饲养效果相当,不存在显著差异（P>0.05）。同时,两性生命表结果表明,人工饲料饲喂的韭蛆平均世代周期23.6800 d,韭菜饲喂的韭蛆世代周期为25.9000 d,两者之间差异显著（P<0.05）。生殖生物学特性分析发现：韭菜和人工饲料饲喂的韭蛆雌成虫净生殖率分别为16.3200和41.1800/d,内禀增长率分别为0.1078和0.1570/d。【结论】筛选的人工饲料和韭菜均可以稳定地完成韭蛆的世代繁殖,且人工饲料饲喂效果略优于韭菜,另外人工饲料较韭菜不但具有低成本省人工的优点,而且可解决韭菜反季节供应和农药残留问题,保证稳定的虫源供给。     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

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。     

Detection of Helicobacter pylori in stool samples of young children using real‐time polymerase chain reaction

Background

The aims of this study were to develop and validate a multiplex real‐time polymerase chain reaction (q‐PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori‐positive samples.

Materials and methods

Archived stool samples from 188 children aged 6‐9 years and 272 samples of 92 infants aged 2‐18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q‐PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q‐PCR and EIA.

Results

Laboratory validation of the q‐PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S‐shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross‐reactivity with other bacterial pathogens was noted. Applying the multiplex q‐PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%‐57%) by q‐PCR (urease cycle threshold <44) vs 59% (95% CI 52%‐66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6‐9 years and 2‐18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.

Conclusions

The developed q‐PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings.