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Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR
实时PCR定量分析昆虫内共生细菌的密度   

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Abstract

Increased attention has been paid to the endosymbiotic bacteria of insects. Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers. Here we report a protocol for real-time qPCR based on SYBR Green I fluorescence as well as some tips to prevent possible pitfalls.

Keywords: Bacteria(细菌), Endosymbionts(内共生体), Insects(昆虫), qPCR(qPCR), Quantification(定量), SYBR Green(SYBR Green)

Background

Insects often harbor bacterial symbionts of various taxa in their bodies. Such bacterial symbionts (endosymbiotic bacteria) attract great attention because of their profound effects on the host insect. Some bacteria provide essential nutrition to their hosts (Baumann et al., 1995), some confer resistance against parasites (Oliver et al., 2003; Hedges et al., 2008), and some even manipulate reproduction or sex determination of their hosts for their own benefit (Werren et al., 2008; Kageyama et al., 2012). Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers (Simoncini et al., 2001), possibly complemented by other visualization methods, such as fluorescence in situ hybridization (FISH) (Koga et al., 2009) and/or electron microscopy.

Materials and Reagents

Note: Reagents differ depending on the qPCR equipment. Here I describe a protocol for absolute quantification using LightCycler® 480 (Roche). For each reaction, two or more technical replicates are strongly recommended.

  1. Pipette tips:
    10-μl tips (e.g., Fukaekasei and Watson, catalog number: 110-201C )
    200-μl tips (Fukaekasei and Watson, catalog number: 1201-705C )
    1,000-μl tips (Fukaekasei and Watson, catalog number: 110-706C )
    To reduce the risk of contamination, use of the filtered tips, e.g.,
    10-μl tips (Fukaekasei and Watson, catalog number: 1251-204CS )
    200-μl tips (Fukaekasei and Watson, catalog number: 1252-703CS )
    1,000-μl tips (Fukaekasei and Watson, catalog number: 124-1000S )] is preferable
  2. Centrifuge tubes of 1.5 ml (e.g., Fukaekasei and Watson, catalog number: 131-7155C )
  3. Centrifuge tubes of 50 ml [e.g., Falcon 50-ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352070 )]
  4. LightCycler® 480 Multiwell Plate 96, white (Roche Molecular Systems, catalog number: 04729692001 , sealing foils included)
  5. Template DNA to be measured (genomic DNA extracted from insects)
  6. Template DNA for standard (PCR products or inserted plasmid DNA with known concentration)
  7. LightCycler® 480, buffer 1 (2x Master mix) (Roche Molecular Systems, catalog number: 04707516001 )
  8. LightCycler® 480, buffer 2 (H2O) (Roche Molecular Systems, catalog number: 04707516001 )
  9. Forward and reverse primers
  10. Reaction mix (see Recipes)

Equipment

  1. Spectrophotometer (Bio-Rad Laboratories, model: SmartSpecTM 3000 )
  2. LightCycler® 480 System (Roche Molecular Systems, model: LightCycler® 480 ) connected to a computer
  3. Centrifuge for 96-well plate (e.g., Kubota, model: PlateSpin II )
  4. Pipettes [e.g., PIPETMAN® P10 (Gilson, catalog number: F144802 ), P20 (Gilson, catalog number: F123600 ), P100 (Gilson, catalog number: F123615 ), P200 (Gilson, catalog number: F123601 ), or P1000 (Gilson, catalog number: F123602 )]
  5. Laminar flow cabinet

Software

  1. LightCycler® 480 Software, version 1.5.1 (Roche Molecular Systems)
  2. Primer3Plus free online software (http://primer3plus.com/)

Procedure

  1. Design primer
    Use a single-copy gene of a target bacterium for primer design (avoid using ribosomal genes because of the possible presence of operonic copies). For example, the bacterial gene RpoB (Case et al., 2007) is considered suitable. If necessary, design primers for the host (insect) gene to standardize the bacterial titers. Use a single-copy nuclear gene from an autosome (mitochondrial genes are not recommended).
    Generally, primers which are 18-23 mer in length are used. The product size should be 80-300 bp (optimum: 80-150 bp). The desired melting temperature (Tm) is 55-65 °C, with a maximum difference of 3 °C in the Tm of the two primers. It is strongly recommended using a software, such as Primer3Plus free online software (http://primer3plus.com/), to design primers.
    Some of the primers that have been successfully used to detect insect endosymbionts are given in Table 1.

    Table 1. Examples of primers for endosymbiotic bacteria that have been successfully used for qPCR


  2. Prepare DNA template
    Extract DNA from the whole body or a particular tissue/organ of the insect. Usually, 50-100 ng of DNA per reaction is needed. The use of purified DNA, such as that extracted using the phenol chloroform method (Green and Sambrook, 2012) or a commercial kit, such as DNeasy® Blood & Tissue Kit (QIAGEN), is recommended. The concentration of the extracted DNA can be measured using a spectrophotometer. Confirm that A260/A280 is approximately 1.8 and A260/A230 is > 1. If possible, DNA extracted from insects treated with antibiotics should also be used as a control, because this will indicate whether the amplified DNA is derived from the bacterial or the host genome (see Note 1 for details).
  3. Prepare standard DNA
    For absolute quantification, DNA samples with known copies of target genes are required. The DNA samples could be PCR products or inserted plasmids. The number of copies can be estimated by mass concentration measured using a spectrophotometer with Avogadro constant NA = 6.022 x 1023 copies/mol, assuming that the molecular weight of 1 bp dsDNA is approximately 660 g/mol. Use a serial dilution of standard DNA, such as 108, 107, 106, 105, 104, and 103 copies.
  4. Prepare the reaction mix (see Recipes), vortex for 5 sec, and dispense 20 μl into each well.
    Apply 5 μl of DNA template (sample or standard DNA) or buffer 2 (negative control) (Figure 1).


    Figure 1. Example of sample sheet. ST: Standard DNA with known concentration, NTC: Non-template control (H2O). When working with two replicates, 41 samples can be measured.

  5. Seal the multiwell plate with sealing foil and centrifuge at 900 x g for 30 sec.
  6. A typical PCR program may be as follows:
    1. Initial denaturation for 30 sec at 95 °C.
    2. Denaturation for 5 sec at 95 °C.
    3. Annealing for 10-30 sec at X °C (X depends on Tm).
    4. Extension for 10-20 sec (depends on product length, 1 min kb-1) at 72 °C.
    5. Return to step (b) for 40 cycles.
    6. Melting at 60 °C-95 °C.
    If the Tm of both the primers is considerably high (> 65 °C), a two-step PCR can be used as follows:
    1. Initial denaturation for 30 sec at 95 °C.
    2. Denaturation for 5 sec at 95 °C.
    3. Annealing and extension for 10-30 s at X °C (X depends on Tm)
    4. Return to step (b) for 40 cycles.
    5. Melting at 60 °C-95 °C.
    Notes:
    1. Data acquisition should be performed at the extension step in each cycle (necessary for generating an amplification curve) and continuously performed at the final melting (necessary for generating a melting curve; Figure 2).
    2. When preparing mixtures, special care should be taken to avoid contamination. It is recommended to use particular pipettes for this purpose in a clean environment, such as in a laminar flow cabinet.
    3. A conserved bacterial gene can be chosen if it is not necessary to classify the bacteria. If the focus is on a specific family, genus, or species, the target gene should be accordingly chosen. Genes that have multiple copies in the genome, such as many outer membrane protein genes, should be avoided. Notably, strain-specific and SNP-containing genes should be avoided because it may compromise the experiment.
    4. When designing primers, the specificity of the primer to the target gene is important. A BLAST search for possible existence of similar nontarget sequences would be helpful for selecting specific primers. It is also desirable that the products be approximately of the same size.

Data analysis

After each reaction, check the melting curve to confirm that the correct products have been amplified. Samples with cycle threshold (Ct) value of > 35 should be considered uninfected or infected at an undetectable level.


Figure 2. Melting curves. Melting curves should be uniform (red lines on the left panel). Two or more patterns (e.g., red and green lines on the right panel) indicate that PCR products are different.

Notes

  1. For detection of any bacterial endosymbiont, it is risky to rely only on PCR and qPCR because of possible pseudo-positives (although most can be identified using melting curves) or the amplification of fragments arising due to horizontal gene transfer (HGT). It is known that many insect species harbor bacterial genome fragments of various sizes in their chromosomes (Dunning Hotopp et al., 2007). Therefore, it is desirable to use multiple target genes to reduce the probability of targeting an HGT gene from the insect genome. If possible, visualize the targeted bacterium using FISH before or after examining the bacterial titers by qPCR. It is helpful to also test insects treated with antibiotics (tetracycline hydrochloride is effective for almost all bacterial species) to demonstrate whether this eliminates/decreases the endosymbiont population.
  2. To standardize the bacterial titers, it is desirable to use the mass concentration of the total genomic DNA as a denominator. However, this may lead to an imprecise estimate of bacterial density because the contribution of bacterial DNA to the total genomic DNA may be significant. For example, Wolbachia pipientis (genome size: ca. 1.2 Mb) whose density is relatively low compared with other obligate endosymbionts, usually yields approximately 10-50 times the number of copies as that of the insect nuclear DNA (Goto et al., 2006). Because the insect genome size is generally 100-300 Mb, bacterial DNA may account for as much as 3.3%-50% of the total genomic DNA, which is too large a proportion to be ignored.
  3. For standardization of bacterial titers, mitochondrial sequence copies are not recommended, because the density of mitochondria can change according to environment, host physiology, and use of antibiotics. Notably, tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila simulans (Ballard and Melvin, 2007).

Recipes

  1. Reaction mix
    In each well (25-μl reaction), add the following reagents:
    Buffer 1 (2x buffer), 12.5 μl
    Buffer 2 (H2O), 2.5 μl
    Primer A (10 μM), 2.5 μl
    Primer B (10 μM), 2.5 μl
    Usually, for one plate (96 wells), a premix can be prepared in a 50-ml tube by adding reagents in the following volumes:
    Buffer 1 (2x buffer), 1,250 μl
    Buffer 2 (H2O), 250 μl
    Primer A (10 μM), 250 μl
    Primer B (10 μM), 250 μl

Acknowledgments

This work was supported by KAKENHI ( 16K08106), Japan.

References

  1. Ballard, J. W. and Melvin, R. G. (2007). Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila. Insect Mol Biol 16(6): 799-802.
  2. Baumann, P., Baumann, L., Lai, C. Y., Rouhbakhsh, D., Moran, N. A. and Clark, M. A. (1995). Genetics, physiology, and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids. Annu Rev Microbiol 49: 55-94.
  3. Case, R. J., Boucher, Y., Dahllöf, I., Holmström, C., Doolittle, W. F. and Kjelleberg, S. (2007). Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol 73: 278-288.
  4. Caspi-Fluger, A., Inbar, M., Mozes-Daube, N., Mouton, L., Hunter, M. S. and Zchori-Fein, E. (2011). Rickettsia ‘in’ and ‘out’: two different localization patterns of a bacterial symbiont in the same insect species. PLoS One 6(6): e21096.
  5. Dunning Hotopp, J. C., Clark, M. E., Oliveira, D. C., Foster, J. M., Fischer, P., Munoz Torres, M. C., Giebel, J. D., Kumar, N., Ishmael, N., Wang, S., Ingram, J., Nene, R. V., Shepard, J., Tomkins, J., Richards, S., Spiro, D. J., Ghedin, E., Slatko, B. E., Tettelin, H. and Werren, J. H. (2007). Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science 317(5845): 1753-1756.
  6. Goto, S., Anbutsu, H. and Fukatsu, T. (2006). Asymmetrical interactions between Wolbachia and Spiroplasma endosymbionts coexisting in the same insect host. Appl Environ Microbiol 72(7): 4805-4810.
  7. Green, M. R. and Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual, Fourth Edition (3-Volume Set). Cold Spring Harbor Laboratory pp: 1890.
  8. Hayashi, M., Watanabe, M., Yukuhiro, F., Nomura, M. and Kageyama, D. (2016). A nightmare for males? a maternally transmitted male-killing bacterium and strong female bias in a green lacewing population. PLoS One 11: e0155794.
  9. Hedges, L. M., Brownlie, J. C., O’Neill, S. L. and Johnson, K. N. (2008). Wolbachia and virus protection in insects. Science 322(5902): 702.
  10. Kageyama, D., Anbutsu, H., Watada, M., Hosokawa, T., Shimada, M. and Fukatsu, T. (2006). Prevalence of non-male-killing spiroplasma in natural populations of Drosophila hydei. Appl Environ Microbiol 72: 6667-6673.
  11. Kageyama, D., Narita, S. and Watanabe, M. (2012). Insect sex determination manipulated by their endosymbionts: incidences, mechanisms and implications. Insects 3(1): 161-199.
  12. Koga, R., Tsuchida, T. and Fukatsu, T. (2009). Quenching autofluorescence of insect tissues for in situ detection of endosymbionts. Appl Entomol Zool 44(2): 281-291.
  13. Narita, S., Nomura, M. and Kageyama, D. (2007). Naturally occurring single and double infection with Wolbachia strains in the butterfly Eurema hecabe: transmission efficiencies and population density dynamics of each Wolbachia strain. FEMS Microbiol Ecol 61: 235-245.
  14. Oliver, K. M., Russell, J. A., Moran, N. A. and Hunter, M. S. (2003). Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci U S A 100: 1803-1807.
  15. Simoncini, L., Casiraghi, M., Bazzocchi, C., Sacchi, L., Bandi, C. and Genchi, C. (2001). Real-time PCR for quantification of the bacterial endosymbionts (Wolbachia) of filarial nematodes. Parassitologia 43(4): 173-178.
  16. Watanabe, M., Yukuhiro, F., Maeda, T., Miura, K. and Kageyama, D. (2014). Novel strain of Spiroplasma found in flower bugs of the genus Orius (Hemiptera: Anthocoridae): transovarial transmission, coexistence with Wolbachia and varied population density. Microb Ecol 67: 219-228.
  17. Werren, J. H., Baldo, L. and Clark, M. E. (2008). Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6: 741-751.

简介

昆虫的内共生菌已被越来越多的关注。 因为大多数昆虫内共生体是不可想象的,所以定量PCR(qPCR)是量子内分泌滴度的实用和方便的方法。 这里我们报告一个基于SYBR Green I荧光的实时qPCR协议,以及一些防止可能的陷阱的提示。
【背景】昆虫通常在其身体中存在各种分类群的细菌共生体。 这种细菌共生体(内共生菌)由于对宿主昆虫的深刻影响而引起了极大的关注。 一些细菌为其宿主提供必要的营养(Baumann等人,1995),一些赋予抗寄生虫的抗性(Oliver等人,2003; Hedges等人 ,2008),有些甚至操纵他们的主机的繁殖或性别决定为自己的利益(Werren等人,2008; Kageyama等人。 ,2012)。 因为大多数昆虫内共生体是不可复制的,所以定量PCR(qPCR)是量子内分泌滴度的实用和方便的方法(Simoncini等人,2001),可能由其他可视化方法补充,例如荧光 >原位杂交(FISH)(Koga等人,2009)和/或电子显微镜。

关键字:细菌, 内共生体, 昆虫, qPCR, 定量, SYBR Green

材料和试剂

注意:试剂因qPCR设备而异。在这里,我描述了使用LightCycler ® 480(Roche)进行绝对定量的协议。对于每个反应,强烈建议使用两个或多个技术重复

  1. 移液器提示:
    10-μl提示(例如,Fukaekasei和Watson,目录号:110-201C)
    200-μl提示(Fukaekasei和Watson,目录号:1201-705C)
    1,000μl提示(Fukaekasei和Watson,目录号:110-706C)
    为了降低污染的风险,请使用过滤的提示,例如,
    10-μl提示(Fukaekasei和Watson,目录号:1251-204CS)
    200-μl提示(Fukaekasei和Watson,目录号:1252-703CS)
    1000μl提示(Fukaekasei和Watson,目录号:124-1000S)]优选
  2. 1.5ml离心管(例如,Fukaekasei和Watson,目录号:131-7155C)
  3. 50毫升例如Falcon 50毫升圆锥形离心管(Corning,Falcon,目录号:352070)]的离心管]
  4. LightCycler ®480 Multiwell Plate 96,white(Roche Molecular Systems,目录号:04729692001,包括密封箔)
  5. 要测量的模板DNA(从昆虫提取的基因组DNA)
  6. 标准DNA(PCR产物或已知浓度的插入质粒DNA)的模板DNA
  7. LightCycler ®480,缓冲液1(2x主混合物)(Roche Molecular Systems,目录号:04707516001)
  8. LightCycler ®480,缓冲液2(H 2 O 2)(Roche Molecular Systems,目录号:04707516001)
  9. 正向和反向引物
  10. 反应混合(见配方)

设备

  1. 分光光度计(Bio-Rad Laboratories,型号:SmartSpec TM 3000)
  2. 连接到计算机的LightCycler ® 480系统(Roche Molecular Systems,型号:LightCycler ® 480)
  3. 离心机用于96孔板(例如,久保田,型号:PlateSpin II)
  4. 移液器[例如,PIPETMAN P10(Gilson,目录号:F144802),P20(Gilson,目录号:F123600),P100(Gilson,目录号:F123615) P200(Gilson,目录号:F123601)或P1000(Gilson,目录号:F123602)]
  5. 层流柜

软件

  1. LightCycler ® 480软件版本1.5.1(Roche Molecular Systems)
  2. Primer3Plus免费在线软件( http://primer3plus.com/

程序

  1. 设计底稿
    使用目标细菌的单拷贝基因进行引物设计(避免使用核糖体基因,因为可能存在操纵子拷贝)。例如,细菌基因RpoB (Case 等人,2007)被认为是合适的。如果需要,设计用于宿主(昆虫)基因的引物来标准化细菌滴度。使用来自自体免疫球蛋白的单拷贝核基因(不推荐线粒体基因) 通常使用长度为18-23的引物。产品尺寸应为80-300bp(最佳值:80-150bp)。所需的熔融温度(T m)为55-65℃,两个引物的T m中的最大差值为3℃。强烈建议您使用一个软件,如Primer3Plus免费在线软件( http://primer3plus.com/ ),设计引物 已经成功用于检测昆虫内共生体的一些引物在表1中给出。

    表1.已成功用于qPCR的内共生菌的引物实例


  2. 准备DNA模板
    从全身或特定的昆虫组织/器官中提取DNA。通常每次反应需要50-100ng的DNA。使用纯化的DNA,例如使用酚氯仿法提取的DNA(Green和Sambrook,2012)或商业试剂盒,例如DNeasy& Blood&推荐使用组织试剂盒(QIAGEN)。提取的DNA的浓度可以使用分光光度计测量。确认A 260> / 280 是大约1.8,而< 260> /< 230>是> 1.如果可能,也应该使用从抗生素处理的昆虫中提取的DNA作为对照,因为这将表明扩增的DNA是否源于细菌或宿主基因组(详见附注1)。
  3. 准备标准DNA
    为了绝对定量,需要具有已知拷贝目标基因的DNA样品。 DNA样品可以是PCR产物或插入的质粒。拷贝数可以通过使用分光光度计测量的质量浓度来估计,其具有Avogadro常数N A = 6.022×10 23 /拷贝/ mol,假设分子量为1b dsDNA约为660g / mol。使用标准DNA的连续稀释液,例如10',10",10",10",10",5" 10 4 和10 3 副本。
  4. 准备反应混合物(参见食谱),旋转5秒,并向每个孔中分配20μl 应用5μlDNA模板(样品或标准DNA)或缓冲液2(阴性对照)(图1)

    图1.样品片的实施例 ST:具有已知浓度的标准DNA,NTC:非模板对照(H 2 O 2)。当进行两次重复时,可以测量41个样品。

  5. 用密封箔密封多孔板,并以900×g离心30秒。
  6. 典型的PCR程序可能如下:
    1. 95℃初始变性30秒
    2. 95℃变性5秒。
    3. 在X°C退火10-30秒(X取决于T )。
    4. 延长10-20秒(取决于产品长度,1分钟kb -1 )在72°C。
    5. 返回步骤(b)40个循环。
    6. 在60°C-95°C熔化。
    如果两个引物的T sub相当高(> 65℃),则可以如下使用两步PCR:
    1. 95℃初始变性30秒
    2. 95℃变性5秒。
    3. X°C退火延伸10-30 s(X取决于T )
    4. 返回步骤(b)40个循环。
    5. 在60°C-95°C熔化。
    注意:
    1. 数据采集应在每个循环的延伸步骤(生成扩增曲线所必需的条件)下进行,并在最终熔化下连续进行(生成熔解曲线所必需的;图2)。
    2. 在制备混合物时,应特别注意避免污染。建议在干净的环境中使用特殊的移液器,例如在层流柜中。
    3. 如果不需要对细菌进行分类,则可以选择保守的细菌基因。如果重点在于特定的家族,属或物种,则应该相应地选择靶基因。应该避免在基因组中具有多个拷贝的基因,如许多外膜蛋白基因。值得注意的是,应避免应变特异性和含SNP的基因,因为它可能危及实验。
    4. 在设计引物时,引物对靶基因的特异性很重要。可能存在类似非目标序列的BLAST搜索将有助于选择特异性引物。也希望产品大致相同的大小。

数据分析

每次反应后,检查解链曲线以确认正确的产品已被放大。循环阈值(Ct)值为> 35应视为未感染或感染不明显的水平。


图2.熔化曲线熔化曲线应均匀(左面板上的红线)。两个或更多个图案(例如右图所示的红色和绿色线)表示PCR产物不同。

笔记

  1. 对于任何细菌内分泌物的检测,由于可能的伪阳性(尽管大多数可以使用解链曲线确定)或由于水平基因转移(HGT)产生的片段的扩增,仅依赖于PCR和qPCR是有风险的。已知许多昆虫物种在其染色体中携带各种大小的细菌基因组片段(Dunning Hotopp et al。,2007)。因此,期望使用多个靶基因降低从昆虫基因组靶向HGT基因的可能性。如果可能,在使用qPCR检查细菌滴度之前或之后,使用FISH观察目标细菌。还可以测试用抗生素治疗的昆虫(四环素盐酸盐对几乎所有细菌物种都有效),以证明这是否消除/减少了内分泌物种群。
  2. 为了标准化细菌滴度,最好使用总基因组DNA的质量浓度作为分母。然而,这可能导致细菌密度的不精确估计,因为细菌DNA对总基因组DNA的贡献可能是显着的。例如,与其他专性内分泌物相比,其密度相对较低的Wolbachia pipientis (基因组大小: 1.2Mb)通常产生约10-50倍的拷贝数如昆虫核DNA(Goto等人,2006))。由于昆虫基因组大小一般为100-300Mb,细菌DNA可能占总基因组DNA的多达3.3%-50%,这个比例太大,不能忽视。
  3. 对于细菌滴度的标准化,不推荐线粒体序列拷贝,因为线粒体的密度可以根据环境,宿主生理学和抗生素的使用而改变。值得注意的是,四环素处理在果蝇模拟物中治疗后二代影响线粒体代谢和mtDNA密度(Ballard和Melvin,2007)。

食谱

  1. 反应混合物
    在每个孔(25μl反应)中,加入以下试剂:
    缓冲液1(2x缓冲液),12.5μl
    缓冲液2(H 2 O 2),2.5μl
    引物A(10μM),2.5μl
    引物B(10μM),2.5μl
    通常,对于一个板(96孔),可以通过以下容积加入试剂在50ml管中制备预混物:
    缓冲液1(2x缓冲液),1,250μl
    缓冲液2(H 2 O 2),250μl
    引物A(10μM),250μl
    引物B(10μM),250μl

致谢

这项工作得到日本的KAKENHI(16K08106)的支持。

参考

  1. Ballard,J.W.and Melvin,R.G。(2007)。 四环素治疗影响治疗后二代的线粒体代谢和mtDNA密度a href ="http://www.ncbi.nlm.nih.gov/pubmed/18093008"target ="_ blank">果蝇 Insect Mol Biol 16(6):799-802。
  2. Baumann,P.,Baumann,L.,Lai,C.Y.,Rouhbakhsh,D.,Moran,N.A。和Clark,M.A。(1995)。 Buchnera属的遗传学,生理学和进化关系:细胞内共生体的蚜虫。 Annu Rev Microbiol 49:55-94。
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  6. Goto,S.,Anbutsu,H。和Fukatsu,T。(2006)。 Wolbachia 和 Spiroplasma 内吞中的不对称互动共存于相同的昆虫宿主中。 Appl Environ Microbiol 72(7):4805-4810。
  7. Green,M.R。和Sambrook,J.(2012)。 分子克隆:实验室手册,第四版(3卷集)冷泉港实验室 pp:1890.
  8. Hayashi,M.,Watanabe,M.,Yukuhiro,F.,Nomura,M。和Kageyama,D。(2016)。 男性的噩梦?一个母亲传播的男性杀伤细菌和一个绿色的辫子人群中的强烈的女性偏见。 PLoS One 11:e0155794。
  9. Hedges,L.M.,Brownlie,J.C.,O'Neill,S.L。和Johnson,K.N。(2008)。 Wolbachia 和昆虫中的病毒防护 科学 322(5902):702.
  10. Kageyama,D.,Anbutsu,H.,Watada,M.,Hosokawa,T.,Shimada,M。和Fukatsu,T。(2006)。 爱因斯坦果蝇自然种群中非雄性杀死性螺旋体的患病率 。 Appl Environ Microbiol 72:6667-6673。
  11. Kageyama,D.,Narita,S.and Watanabe,M。(2012)。 由他们的内在信息操纵的昆虫性别决定:发生,机制和影响。 昆虫 3(1):161-199。
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  13. Narita,S.,Nomura,M。和Kageyama,D。(2007)。 蝴蝶中的自然发生的单次和双重感染Wolbachia 菌株> Eurema hecabe :每种Wolbachia菌株的传播效率和种群密度动态。 FEMS Microbiol Ecol 61:235-245。
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引用:Kageyama, D. (2017). Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR. Bio-protocol 7(19): e2566. DOI: 10.21769/BioProtoc.2566.
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