Ampdirect^® Gene Amplification

新型 PCR 扩增缓冲液

　　——无需抽提 DNA，直接 PCR 扩增！

　　Ampdirect^® Gene Amplification 是一款新开发的 PCR 缓冲液，可有效降低 PCR 反应体系里的蛋白质、糖类等各种 PCR 抑制物对 PCR 扩增的影响。应用广泛，适合对昆虫、植物、微生物、土壤、血液、石蜡切片等各类样品直接进行 PCR 扩增。

◆优势

●　省时省力省钱

　  无需 DNA 抽提，直接进行 PCR 反应，节省时间

●　适用于微量样品

　  因为不需要 DNA 抽提，不会产生样品损失，故此十分适用于微量样品

●　热启动酶

　  厂家自行开发的高性能聚合酶 BIOTAQ ™

●　支持后续实验操作

　  扩增产物可用于后续的测序和 RFLP 等片段分析

◆实验流程

◆原理

◆示例

注：动物样本，比如血液和黏膜细胞等可以直接加入 PCR 反应液，不需消化处理。固体样本，比如植物和动物组织需要在含有 SDS 和蛋白酶 K 的

       消化液中处理后再进行 PCR 反应，或者使用 FTA 卡收集植物组织中的 DNA，取带有 DNA 的 FTA 卡加入 PCR 反应液进行PCR 反应。

◆其他应用

● 血清中病毒检出

● 小鼠尾巴裂解液检测基因分型

● 植物、血液/ 滤纸血液、土壤

● 昆虫、石蜡切片等微量样品

   其他应用的详细内容请看相关资料！

相关资料

◆Ampdirect 推荐使用酶与不推荐使用酶

◆其他应用

Ampdiret 相关的FAQ

◆有关样品以及样品前处理

1．采集后长期保存的血液作为模板，能进行 PCR 吗？

实验证明，采集后冻存了长达五年时间的血或纸血（干燥血）作为模板，依然能进行PCR。必须长期稳定保存血液时，可使用 Whatman 公司的 FTA Card（核酸保存用卡）进行保存。关于血液（全血）对 PCR 的抑制作用，直接采集的血液（新鲜血）对 PCR 抑制作用更强。

另外，长久以来的经验证明，采用干燥血作为 PCR 模板时，对比用一般的实验纸保存，使用 FTACard 保存的血液进行 PCR 的结果更稳定，因此推荐使用 FTA Card 保存的血作为 PCR 模板。

2．从粪便中抽提的 DNA 模板，可用 PCR 检测出细菌吗？

粪便抽提的 DNA 对 PCR 有抑制作用，因此用稀释后的 DNA 反应。从粪便样品中简便进行 PCR 的应用例子，在“使用实时 PCR 法高灵敏度检测 Vero Toxin”中有介绍。将10%的粪便悬浊液热处理（95℃，5 分钟）后，将离心后的上清液作为 PCR 模板。实验证明，使用这个方法，大部分的粪便样品都能稳定进行 PCR 。

3．Ampdirect 可应用于 RT-PCR 吗？粪便里存在的 RNA 病毒也可以检测出来吗？

从粪便样品中简便检测 RNA 病毒的应用例子，可见“粪便中的 RNA 病毒检测”。将粪便悬浊液离心后的上清液，涂布在 Whatman 公司的 FTA Card（核酸保存用卡）上，干燥后，在卡上打（φ1.25 mm）就可作为模板使用。

Ampdirect Plus 可做为逆转录反应的反应液，逆转录酶选用的是 Invitrogen 公司的 M-MLVReverse Transcriptase（Cat No. 28025-013）。

4．样品的溶解处理最后步骤要在95℃，进行５分钟的热处理，这个热处理步骤是必须的吗？

因为要使溶解液中的 Proteinase K 失活，所以必须热处理。如果不进行热处理，Proteinase K 仍保持活性的话，在往 PCR 反应液里加入溶解液时，Proteinase K 会将 PCR 酶（Taq DNA Polymerase）降解掉，从而不能进行稳定的 PCR。实际上，这是从大部分得不到目的 PCR 产物的例子上取得的经验。所以，必须在最后对溶解液进行热处理（95℃，5 分钟）。

5．从植物的叶片抽提的 DNA 作为 PCR 模板进行反应，但得不到目的 PCR 产物。考虑到是植物里所含的糖类抑制了 PCR，使用 Ampdirect 可以改善这个情况吗？

至今为止，已有非常多客户反映，从实验植物、谷物、蔬菜、果蔬的叶片取样，用 Ampdirect 进行 PCR，都取得了良好的结果。不同植物对 PCR 的抑制作用也不尽相同，可参照“从植物取样的简便 PCR 实例”。在 PCR 前用溶解液进行前处理。如担心因为 PCR 被抑制而得不到目的 PCR 产物，用溶解处理液稀释５~10 倍，会对此情况有很大改善。

6．小鼠尾部用溶解液溶解了大约１个小时，还没完全溶解。可作为 PCR 的模板使用吗？

将小鼠尾巴１~５mm 浸入 100 μL 溶解液内，55℃下溶解约１小时后，如小鼠尾巴还未完全溶解，对 Proteinase K 进行失活处理（95℃，5 分钟）后，从溶解液中提取出来的 DNA 量（拷贝数）应该足以作为 PCR 模板，大部分情况下进行 PCR 没有问题。

与此对照，如果是将大条的小鼠尾巴（５ mm 以上）过夜处理以至完全溶解，溶解液会变成高粘度状态。这种情况下，过剩的 DNA 反而会抑制 PCR 反应，请用蒸馏水或 TE 将溶解液稀释 10 倍左右。请注意尽量不要让小鼠尾巴过度溶解。

另外，溶解处理后的溶解液不需离心，只需取上清液就可作为 PCR 模板使用。从以往经验得知，经溶解处理过的溶解液在冷藏保存条件下保存２~３年内皆可作为 PCR 模板使用。

7．不对唾液进行前处理就直接作为 PCR 模板是不可行的吧？“取样自口腔粘膜的 PCR”这个应用，是对口腔黏膜细胞用溶解液进行溶解处理，如果是用唾液的话，也必须用溶解液进行溶解处理吗？

目前，也有不少客户为了检测唾液中的微生物，不进行前处理，直接将唾液当成 PCR 模板使用。当作为模板进入 PCR 反应液的唾液中（数 μL）有大量微生物，可获得良好的 PCR 产物。与之相反，若微生物数量较少，根据唾液中的蛋白等物质对 PCR 抑制的不同程度，有可能得不到稳定的 PCR 产物。因此，推荐对溶液用溶解液进行溶解处理，再作为 PCR 模板。对唾液进行了溶解处理的话，可得到以下两种结果，①“抽提微生物的DNA”和②“降低唾液对 PCR 的抑制作用（分解唾液中的蛋白）”，也可得到稳定的 PCR 结果。

8．如何用 PCR 检测血浆、血清样品中的微生物？

血浆、血清这些全血，对PCR 有较强抑制作用，所以不能将全血直接用作 PCR 模板。因此，要用于“检测血清中的病毒”时，将血浆、血清用溶解液进行溶解处理，分解掉血浆、血清中的蛋白，降低对PCR 的抑制作用。

请注意，本方法适用于检测 DNA 病毒、细菌，不可用于检测 RNA 病毒。检测 RNA 病毒时，在溶解处理步骤，因为血浆、血清中存在 RNA 分解酶，从病毒外壳裸露出来的 RNA 就会被分解。

9、使用 FTA Card 和实验纸作为模板进行多样品 PCR 时，因为使用打孔器等工具可能会引起污染。有没有相应的对策呢？

采集血液样品时，首次打孔取样后，在下一次取样前，用打孔器在 FTA Card 或实验纸上没有血样的空白处空打３个孔，这样可保证打孔器上不会残留前一样品的模板（血样是白血球 DNA）。因为从客户处得知，“每次用打孔器取样时，用金伯利（Kimwipe）浸渍酒精擦拭打孔器的打孔部分，这个方法反而引起污染”，所以推荐空打孔的方法。但是，检测拷贝数多的 DNA，如线粒体 DNA 和质粒 DNA 等时，本方法不能防止污染。

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Indian Journal of Science and Technology Vol.2 No. 10 (Oct 2009)

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样品：植物（拟南芥）              

Plant Biotechnology 26(4), 435-441, 2009

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样品：植物（烟草）           

Genes Brain Behav. 2009 Oct;8(7):650-60.

・    A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy.

　    Suzuki H, et al.

样品：大鼠           

J Plant Res 2009; 122: 585-95

・    Genetic population structure of Osmunda japonica, rheophilous Osmunda lancea and their hybrids

　    Yatabe Y, et al.

样品：植物           

Am J Physiol Renal Physiol 2009; 297: F671-8

・    Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development

　    Segawa H, et al.

样品：小鼠           

J Immunol 2009;183: 3053-63

・    Crucial contribution of thymic Sirp alpha+ conventional dendritic cells to central tolerance against blood-borne antigens in a CCR2-dependent manner

　    Baba T, et al.

样品：小鼠           

J Nat Med 2009; 63: 340-4

・    The botanical origin of kratom (Mitragyna speciosa; Rubiaceae) available as abused drugs in the Japanese markets

　    Maruyama T, et al.

样品：植物         

分析手法：PCR-RFLP法          

Emerg Infect Dis 2009;15: 912-5

・    Bartonella quintana in body lice and head lice from homeless persons, San Francisco, California, USA

　    Bonilla DL, et al.

样品：虱子、細菌              

Plant Cell Physiol 2009; 50: 1579-86

・    Arabidopsis bile acid:sodium symporter family protein 5 is involved in methionine-derived glucosinolate biosynthesis

　    Sawada Y, et al.

样品：植物（拟南芥）              

Mol Genet Metab 2009; 97: 190-5

・    High frequency of acid alpha-glucosidase pseudodeficiency complicates newborn screening for glycogen storage disease type II in the Japanese population

　    Kumamoto S, et al.

样品：滤纸血        

分析手法：ARMS-PCR法         

Laboratory Animal research 2009; 25: 75-8

・    Simple Genotyping Method Using Ampdirect Plus and FTA Technologies: Application to the Identification of Transgenic Animals and Their Routine Genetic Monitoring

　    Nakanishi S, et al.

样品：小鼠-大鼠、FTA card              　

Int. J. Environ. Res. Public Health 2009; 6: 999-1009

・    Association between a Polymorphism of Aminolevulinate Dehydrogenase (ALAD) Gene and Blood Lead Levels in Japanese Subjects

　    Miyaki K, et al.

样品：人全血              

Jpn J Infect Dis 2009; 62: 164-7

・    Direct Colony PCR of Several Medically Important Fungi using Ampdirect(R) Plus

　    Alshahni MM, et al.

样品：真菌（酵母・霉菌）              

Biochem. Eng J 2009; 45: 76-81

・    Characterization of bacterial population of raw milk from bovine mastitis by culture-independent

PCR-DGGE method

　    Kuang Y, et al.

样品：牛乳、細菌     

分析手法：PCR-DGGE法          　

Lab Chip 2009; 9: 1052-8

・    Microfluidic device using chemiluminescence and a DNA-arrayed thin film transistor photosensor for single nucleotide polymorphism genotyping of PCR amplicons from whole blood

　    Hatakeyama K, et al.

样品：人全血      

分析手法：PCR-RFLP法          

Cancer Cell 2009; 15: 195-206

・    Cancer metastasis is accelerated through immunosuppression during Snail-induced EMT of cancer cells

　    Kudo-Saito C, et al.

Vet Microbiol 2009; 135: 261-6

・    Detection of cyprinid herpesvirus 3 DNA in river water during and after an outbreak

　    Minamoto T, et al.

样品：河川水、DNA病毒         

Methods Mol Biol 2009; 538: 7-27

・    Backtracking of leukemic clones to birth

　    Wiemels J, et al.

样品：新生儿滤纸血           

Int J Plant Sci 2009; 170: 237-46

・    Phylogenetic relationship and morphology of Dalzellia gracilis (Podostemaceae, subfamily Tristichoideae)

with proposal of a new genus

　    Koi S, et al.

样品：植物           

Infect Dis Clin Pract 2008; 16: 230-4

・    Association of the SLC11A1 Gene Polymorphisms With Susceptibility to Micobacterium Infections in a Japanese Population

　    Asai S, et al.

J Vet Diagn Invest 2008; 20: 68-71

・    Molecular screening of canine GM1 gangliosidosis using blood smear specimens after prolonged storage:

detection of carriers among shiba dogs in northern Japan

　    Yamato O, et al.

Biosci Biotechnol Biochem 2008; 72: 2831-9

・    Diversity and Similarity of Microbial Communities in Petroleum Crude Oils Produced in Asia

　    Yamane K, et al.

Vector Borne Zoonotic Dis 2008; 8: 565-73

・    Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies

　    Konnai S, et al.

J. Pestic Sci 2008; 33: 122-7

・    Environmental distribution and novel high-throughput screening of APEO-degrading bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS)

　    Ichiki Y, et al.

Zootaxa 2008; 1746: 15-38

・    Molecular systematics and biogeography of the genus Zizina(Lepidoptera: Lycaenidae)

　    Yago M, et al.

Extremophiles 2008; 12: 519-27

・    Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula

　    Kobayashi T, et al.

Principles and Technical Aspects of PCR Amplification

・    pp91-101, Springer Netherlands, 2008

　    Elizabeth van Pelt-Verkuil, et al.

Zoolog Sci 2008; 25: 838-42

・  Phylogenetic Position of the Endemic Large Carpenter Bee of the Ogasawara Islands, Xylocopa ogasawarensis (Matsumura, 1912) (Hymenoptera: Apidae), Inferred from Four Genes

　    Kawazoe K, et al.

Mol Phylogenet Evol 2008; 49: 503-13

・    Redundant species, cryptic host-associated divergence, and secondary shift in Sennertia mites (Acari: Chaetodactylidae) associated with four large carpenter bees (Hymenoptera: Apidae: Xylocopa) in the Japanese island arc

　    Kawazoe K, et al.

Odonatologica 2008; 37: 131-44

・    Population genetic differentiaiton in three sympatric damselfly species in a highly fragmented urban landscape

　    Sato M, et al.

Blood 2008; 111: 376-8

・    NOTCH1 mutation can be an early, prenatal genetic event in T-ALL

　    Eguchi-Ishimae M, et al.

Res Vet Sci 2007; 82: 54-60

・    Nonsense mutation of feline beta-hexosaminidase beta-subunit (HEXB) gene causing Sandhoff disease in a family of Japanese domestic cats

　    Kanae Y, et al.

Leuk Res 2007; 31: 1633-40

・    Regulatory polymorphisms of multidrug resistance 1 (MDR1) gene are associated with the development of childhood acute lymphoblastic leukemia

　    Hattori H, et al.

Microbiol Immunol 2007; 51: 507-17

・    Plasma levels of unactivated thrombin activatable fibrinolysis inhibitor (TAFI) are down-regulated in young

adult women: analysis of a normal Japanese population

　    Akatsu H, et al.

Am J Botany 2007; 94: 1630-41

・    Cryptic species and host specificity in the ectomycorrhizal genus Strobilomyces (Strobilomycetaceae)

　    Sato H, et al.

Botanical Journal of the Linnean Society 2007; 155, 1-27

・    A global molecular phylogeny of the fern genus Trichomanes (Hymenophyllaceae) with special reference to stem anatomy

　    Ebihara A, et al.

Trop Anim Health Prod 2007; 39: 369-74

・    Comparison of polymerase chain reaction methods for the detection of Theileria equi infection using whole blood compared with pre-extracted DNA samples as PCR templates

　    Alhassan A, et al.

Am J Trop Med Hyg 2007; 77: 324-9

・    Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods.

　    Kato H, et al

Metabolism 2006; 55: 751-7

・    G-protein beta 3 subunit polymorphism C1429T and low-density lipoprotein receptor-related protein 5 polymorphism A1330V are risk factors for hypercholesterolemia in Japanese males--a prospective study over 5 years

　    Suwazono Y, et al.

J Vet Med Sci 2006; 68: 27-33.

・    Sequence variation of bovine prion protein gene in Japanese cattle (Holstein and Japanese Black)

　    Nakamitsu S, et al.

Ann Hum Genet 2006; 70: 767-77

・    G-protein beta3 subunit variant C825T is a risk factor for hypertension in Japanese females -a prospective cohort study over 5 years

　    Suwazono Y, et al.

J Biosci Bioeng 2006; 102: 572-4

・    High-throughput genotyping of filamentous fungus Aspergillus oryzae based on colony direct polymerase chain reaction.

　    Suzuki S, et al

Biosci Biotechnol Biochem 2006; 70: 2387-93

・    The molecular phylogeny of the genus Rhizopus based on rDNA sequences.

　    Abe A, et al

Appl Environ Microbiol 2006; 72: 7912-5

・    Molecular detection of epiphytic Acaryochloris spp. on marine macroalgae.

　    Ohkubo S, et al

Tohoku J Exp Med 2006; 209: 149-57

・    G-protein beta3 subunit gene variant is unlikely to have a significant influence on serum uric acid level in Japanese workers.

　    Suwazono Y, et al

Ornithol Sci 2006; 5: 139-143

・    Usefulness of avian buccal cells for molecular sexing.

　    Arima H, et al

Int J Mol Med 2006; 17: 77-82

・    The -1438A/G polymorphism in the 5-hydroxytryptamine receptor 2A gene is related to hyperuricemia, increased gamma-glutamyl transpeptidase and decreased high-density lipoprotein cholesterol level in the Japanese population: a prospective cohort study over 5 years.

　    Suwazono Y, et al

Arch Virol 2005; 150: 1927-31

・    Rotavirus antigenemia in children with encephalopathy accompanied by rotavirus gastroenteritis

　    Nakagomi T, et al.

Thromb Res 2005; 115: 191-7

・    Factor XII Shizuoka, a novel mutation (Ala392Thr) identified and characterized in a patient with congenital coagulation factor XII deficiency

　    Oguchi S, et al.

Hemoglobin 2005; 29: 1-10

・     Hb KOCHI [beta141(H19)Leu-->Val (g.1404C-->G); 144-146(HC1-3)Lys-Tyr-His -->0 (g.1413 A-->T)]: a new variant with increased oxygen affinity

　    Miyazaki A, et al.

J Epidemiol 2005; 15: 203-10

・     Increased risk of obesity resulting from the interaction between high energy intake and the Trp64Arg polymorphism of the beta3-adrenergic receptor gene in healthy Japanese men.

　    Miyaki K, et al

Br J Pharmacol 2005; 145: 818-28

・     Inhibition of the antigen-induced activation of rodent mast cells by putative Janus kinase 3 inhibitors WHI-P131 and WHI-P154 in a Janus kinase 3-independent manner.

　    Watchara Linwong, et al

Blood Coagul Fibrinolysis 2004; 15: 367-73

・    Genetic analyses and expression studies identified a novel mutation (W486C) as a molecular basis of congenital coagulation factor XII deficiency

　    Ishii K, et al.

Genes Chromosomes Cancer 2004; 39: 335-40

・    Protracted postnatal natural histories in childhood leukemia

　    Maia AT, et al.

Obes Res 2004; 12: 4-8

・     Lack of association between human G-protein beta3 subunit variant and overweight in Japanese workers.

　    Suwazono Y, et al

Drug Matab. Pharmacokin. 2004; 19: 303-7

・    Genotyping of single nucleotide polymorphism (SNPs) influencing drug response by competitive allele-specific short oligonucleotide hybridization (CASSOH) with immunochromatographic strip.

　    Hiratsuka M, et al

J Vet Diagn Invest 2004; 16: 469-72

・    Rapid and simple mutation screening of GM1 gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

　    Yamato O, et al

Blood Coagul Fibrinolysis 2003; 14: 663-70

・    Association of the -159 C --> T polymorphism in the CD14 promoter with variations in serum lipoproteins in healthy subjects.

　    Eilertsen KE, et al.

GENES, Chromosomes & Cancer 2003; 37: 36-43

・    Prenatal origin of TEL-AMLI-positive acute lymphoblastic leukemia in children born in California.

　    McHale CM, et al

Arch Virol 2002; 147: 2187-95

・    Molecular characterization of serotype G2 and G3 human rotavirus strains that have an apparently identical electropherotype of the short RNA pattern

　    Nakagomi T, et al.

BLOOD；2002；99：3801-5

・    In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia

　    Wiemels JL, et al

Proc. Natl. Acad. Sci. USA 2002; 99: 15101-6

・    Site-specific translocation and evidence of postnatal origin of the t(1;19) E2A-PBX1 fusion in childhood acute lymphoblastic leukemia.

　    Wiemels JL, et al

Clin Lab; 2002; 48: 377-84

・    Various applications of direct PCR using blood samples

　    Nishimura N, et al

Proceedings of the Seventh International Colloquium on Paratuberculosis; 2002; 267-9

・    Efficiency of polymerase chain reaction using a novel method of DNA preparation and amplification for detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples

　    Kojima K, et al

Arch Virol 2001; 146: 557-70

・    Direct evidence for genome segment reassortment between concurrently-circulating human rotavirus strains

　    Watanabe M, et al.

Transfusion 2000; 40: 1081-7

・    Elimination of both cell-free and cell-associated HIV infectivity in plasma by a filtration/methylene blue photoinactivation system

　    Abe H, et al.

Microbiol Immunol 2000; 44: 957-61

・    Apparent re-emergence of serotype G9 in 1995 among rotaviruses recovered from Japanese children hospitalized with acute gastroenteritis

　    Oka T, et al

J Clin Microbiol 2000; 38: 2649-54

・    Major change in the predominant type of “Norwalk-like viruses” in outbreaks of acute nonbacterial gastroenteritis in Osaka city, Japan, between April 1996 and March 1999

　    Iritani N, et al

Blood; 2000; 96:264-8

・    Detection of clonotypic IGH and TCR rearrangements in the neonatal blood spots of infants and children with B-cell precursor acute lymphoblastic leukemia

　    Yagi T, et al

Ann Clin Biochem; 2000; 37: 674-80

・    Direct PCR from whole blood without DNA isolation

　    Nishimura N, et al

Stroke 2000; 31: 2661-4

・    Polymorphism in the promoter of lipopolysaccharide receptor CD14 and ischemic cerebrovascular disease

　    Ito D, et al

Stroke 2000; 31: 936-9

・     C242T polymorphism of NADPH oxidase p22 PHOX gene and ischemic cerebrovascular disease in the Japanese population

　    Ito D, et al

Stroke 2000; 31: 493-7

・    Association between platelet glycoprotein Iba genotype and ischemic cerebrovascular disease

　    Sonoda A, et al