Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

Plant Methods

Chunqing Zhang¹  Daxing Wen¹ 

[1] State Key Laboratory of Crop Biology, Agronomy College, Shandong Agricultural University, Tai’an, Shandong Province 271018, P. R. China

关键词: Maize;    Genetic purity;    Species;    Polymorphisms;    Multiplex PCR;   

Others  :  821788
DOI  :  10.1186/1746-4811-8-32

 received in 2012-07-13, accepted in 2012-07-25,  发布年份 2012

【 摘 要 】

Background

Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality.

Results

A novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed “Two Rounds Mode” (three and 28–32 cycles). The first round (the first three cycles) is named the “One by One Annealing Round”. The second round (28–32 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round.

Conclusions

UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species.

【 授权许可】

   
2012 Wen and Zhang; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Choudhary R, Paliwal J, Jayas DS: Classification of cereal grains using wavelet, morphological, colour, and textural features of non-touching kernel images. Biosyst Eng 2008, 99(3):330-337.
• [2]Hoffman D, Hang A, Larson S, Jones B: Conversion of an RAPD marker to an STS marker for barley variety identification. Plant Mol Biol Rep 2003, 21(1):81-91.
• [3]Hulya I: RAPD markers assisted varietal identification and genetic purity test in pepper, Capsicum annuum. Sci Hortic-Amsterdam 2003, 97(3):211-218.
• [4]Liu L, Wang Y, Gong Y, Zhao T, Liu G, Li X, Yu F: Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers. Sci Hortic-Amsterdam 2007, 115(1):7-12.
• [5]Liu L, Liu G, Gong Y, Dai W, Wang Y, Yu F, Ren Y: Evaluation of Genetic Purity of F1 Hybrid Seeds in Cabbage with RAPD, ISSR, SRAP, and SSR Markers. Hortscience 2007, 42(3):724-727.
• [6]Macko A, Grzebelus D: DcMaster transposon display markers as a tool for diversity evaluation of carrot breeding materials and for hybrid seed purity testing. J Appl Genet 2008, 49(1):33-39.
• [7]Massaux C, Sindic M, Lenartz J, Sinnaeve G, Bodson B, Falisse A, Dardenne P, Deroanne C: Variations in physicochemical and functional properties of starches extracted from European soft wheat (Triticum aestivum L.): The importance to preserve the varietal identity. Carbohyd Polym 2008, 71(1):32-41.
• [8]Naresh V, Yamini K, Rajendrakumar P, Dinesh Kumar V: EST-SSR marker-based assay for the genetic purity assessment of safflower hybrids. Euphytica 2009, 170(3):347-353.
• [9]Pattemore JA, Rice N, Marshall DF, Waugh R, Henry RJ: Cereal variety identification using MALDI-TOF mass spectrometry SNP genotyping. J Cereal Sci 2010, 52(3):356-361.
• [10]Pranavi B, Sitaram G, Yamini KN, Kumar VD: Development of EST-SSR markers in castor bean (Ricinus communis L.) and their utilization for genetic purity testing of hybrids. Genome 2011, 54:684-691.
• [11]Sundaram R, Naveenkumar B, Biradar S, Balachandran S, Mishra B, IlyasAhmed M, Viraktamath B, Ramesha M, Sarma N: Identification of informative SSR markers capable of distinguishing hybrid rice parental lines and their utilization in seed purity assessment. Euphytica 2008, 163(2):215-224.
• [12]Terzi V, Morcia C, Gorrini A, Stanca AM, Shewry PR, Faccioli P: DNA-based methods for identification and quantification of small grain cereal mixtures and fingerprinting of varieties. J Cereal Sci 2005, 41(3):213-220.
• [13]Wiwart M, Suchowilska E, Lajszner W, Graban Ł: Identification of hybrids of spelt and wheat and their parental forms using shape and color descriptors. Comput Electron Agr 2012, 83:68-76.
• [14]Wu M, Jia X, Tian L, Lv B: Rapid and Reliable Purity Identification of F1 Hybrids of Maize (Zea may L.) Using SSR Markers. Mol, Plant Breeding 2006, 3(4):381-384.
• [15]Zhou X, Shen S, Wu D, Sun J, Shu Q: Introduction of a xantha mutation for testing and increasing varietal purity in hybrid rice. Field Crop Res 2006, 96(1):71-79.
• [16]Bacchetta G, García PE, Grillo O, Mascia F, Venora G: Seed image analysis provides evidence of taxonomical differentiation within the Lavatera triloba aggregate (Malvaceae). Flora 2011, 206(5):468-472.
• [17]Chen X, Xun Y, Li W, Zhang J: Combining discriminant analysis and neural networks for corn variety identification. Comput Electron Agr 2010, 71(Supplement 1(0)):S48-S53.
• [18]Dubey BP, Bhagwat SG, Shouche SP, Sainis JK: Potential of Artificial Neural Networks in Varietal Identification using Morphometry of Wheat Grains. Biosyst Eng 2006, 95(1):61-67.
• [19]Granitto PM, Verdes PF, Ceccatto HA: Large-scale investigation of weed seed identification by machine vision. Comput Electron Agr 2005, 47(1):15-24.
• [20]Manickavasagan A, Sathya G, Jayas DS: Comparison of illuminations to identify wheat classes using monochrome images. Comput Electron Agr 2008, 63(2):237-244.
• [21]Medina W, Skurtys O, Aguilera JM: Study on image analysis application for identification Quinoa seeds (Chenopodium quinoa Willd) geographical provenance. LWT- Food Sci Technol 2010, 43(2):238-246.
• [22]Yan X, Liu S, Zhang C, Wang J: Purity identification of maize seed based on color characteristics. Transactions CSAE 2010, 26:46-50.
• [23]Granitto PM, Navone HD, Verdes PF, Ceccatto HA: Weed seeds identification by machine vision. Comput Electron Agr 2002, 33(2):91-103.
• [24]Smith JSC, Chin ECL, Shu H, Smith OS, Wall SJ, Senior ML, Mitchell SE, Kresovich S, Ziegle J: An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPS and pedigree. Theor Appl Genet 1997, 95(1):163-173.
• [25]Armani A, Castigliego L, Tinacci L, Gianfaldoni D, Guidi A: Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh, marinated and cooked products. Food Chem 2012, 133(1):184-192.
• [26]Bai W, Xu W, Huang K, Yuan Y, Cao S, Luo Y: A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species. Food Control 2009, 20(4):366-370.
• [27]Chang Y, Yeh K, Hsu NC, Lin S, Chang T, Chang J: Detection of N-, H-, and KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-specific primer extension. Clin Biochem 2010, 43(3):296-301.
• [28]Espiñeira M, Atanassova M, Vieites JM, Santaclara FJ: Validation of a method for the detection of five species, serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood. Food Microbiol 2010, 27(1):122-131.
• [29]Gunson R, Maclean A, Davies E, Bennett S, Miller R, Carman WF: Development of a multiplex real-time RT-PCR that allows universal detection of influenza A viruses and simultaneous typing of influenza A/H1N1/2009 virus. J Virol Methods 2010, 163(2):258-261.
• [30]Hu Q, Tu J, Han X, Zhu Y, Ding C, Yu S: Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks. J Microbiol Meth 2011, 87(1):64-69.
• [31]Ao J, Li Q, Gao X, Yu Y, Li L, Zhang M: A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean, maize and rice in highly processed products. Food Control 2011, 22(10):1617-1623.
• [32]Khamrin P, Okame M, Thongprachum A, Nantachit N, Nishimura S, Okitsu S, Maneekarn N, Ushijima H: A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea. J Virol Methods 2011, 173(2):390-393.
• [33]Kim H, Lee H, Lee K, Cho J: Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR. Food Control 2012, 23(2):491-498.
• [34]Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S: Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers. J Microbiol Meth 2011, 86(1):119-120.
• [35]Langrell SRH, Morel O, Robin C: Touchdown nested multiplex PCR detection of Phytophthora cinnamomi and P. cambivora from French and English chestnut grove soils. Fungal Biol 2011, 115(7):672-682.
• [36]Lu I, Lin C, Pan T: Establishment of a system based on universal multiplex-PCR for screening genetically modified crops. Anal Bioanal Chem 2010, 396(6):2055-2064.
• [37]Milheirico C, Oliveira DC, de Lencastre H: Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: ‘SCCmec IV multiplex’. J Antimicrob Chemother 2007, 60(3):42-48.
• [38]Pichon J, Bonnaud B, Cleuziat P, Mallet F: Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling. Nucleic Acids Res 2006, 34(6):e46.
• [39]Rugman-Jones P, Forster L, Guerrieri E, Luck R, Morse J, Monti M, Stouthamer R: Taxon-specific multiplex-PCR for quick, easy, and accurate identification of encyrtid and aphelinid parasitoid species attacking soft scale insects in California citrus groves. Biocontrol 2011, 56(3):265-275.
• [40]Sankuntaw N, Sukprasert S, Engchanil C, Kaewkes W, Chantratita W, Pairoj V, Lulitanond V: Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system. Mol Cell Probe 2011, 25(2–3):114-120.
• [41]Shigemori Y, Mikawa T, Shibata T, Oishi M: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products. Nucleic Acids Res 2005, 33(14):e126.
• [42]Wang C, Jong Y, Chang J, Chen Y, Wu S: Universal fluorescent multiplex PCR and capillary electrophoresis for evaluation of gene conversion between SMN1 and SMN2 in spinal muscular atrophy. Anal Bioanal Chem 2010, 397(6):2375-2383.
• [43]Wang R, Huang J, Zhang W, Lin G, Lian J, Jiang L, Lin H, Wang S, Wang S: Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA–DNA hybridization on a microarray. J Genet Genomics 2011, 38(3):129-135.
• [44]Xu W, Bai W, Luo Y, Yuan Y, Zhang W, Guo X, Huang K: A novel common single primer multiplex polymerase chain reaction (CSP-M-PCR) method for the identification of animal species in minced meat. J Sci Food Agr 2008, 88(15):2631-2637.
• [45]Yuan Y, Xu W, Zhai Z, Shi H, Luo Y, Chen Z, Huang K: Universal primer-multiplex PCR approach for simultaneous detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in food samples. J Food Sci 2009, 74(8):M446-M452.
• [46]Dehainault C, Lauge A, Caux-Moncoutier V, Pages-Berhouet S, Doz F, Desjardins L, Couturier J, Gauthier-Villars M, Stoppa-Lyonnet D, Houdayer C: Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene. Nucleic Acids Res 2004, 32(18):e139.
• [47]Chamberlain J, Gibbs R, Ranier J, Nguyen P, Caskey C: Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988, 16(23):11141-11156.
• [48]Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M: AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 1995, 23(21):4407-4414.