- 一、真菌的分子生物学鉴定
早期病原真菌的分子生物学鉴定仅利用真菌rDNA的核糖体内转录间隔区（ITS）分子标记，快速鉴定病原真菌。但随后的研究发现，ITS序列不能将有些真菌属内不同种精确的区分开，学者进一步开发出基于多基因的系统发育分析方法。
目前刺盘孢属真菌种类鉴定普遍以 ITS、肌动蛋白 (ACT)、钙调蛋白(CAL)、3-磷酸甘油醛脱氢酶 (GAPDH)、几丁质合成酶 A 基因(CHS-I)和B-微管蛋白(TUB2) 等多基因进行系统发育分析
| Gene | Primer | Sequence(5’-3’) | Reference |
| ITS | ITS1 | TCCGTAGGTGAACCTGCGG | Gardes and Bruns 1993 |
| ITS4 | TCCTCCGCTTATTGATATGC | ||
| ACT | ACT-512 | ATGTGCAAGGCCGGTTTCGC | Carboneand Kohn 1999 |
| ACT-783R | TACGAGTCCTTCTGGCCCAT | ||
| CAL | CAL-228F | GAGTTCAAGGAGGCCTTCTCCC | Carboneand Kohn 1999 |
| CAL-737R | CATCTTTCTGGCCATCATGG | ||
| GAPDH | GDF1 | GCCGTCAACGACCCCTTCATT | Guerber et al 2003 |
| GDR2 | GGGTGGAGTCGTACTTGAGCATGT | ||
| CHS-I | CHS-79F | TGGGGCAAGGATGCTTGGAAGAAG | Carboneand Kohn 1999 |
| CHS-354R | TGGAAGAACCATCTGTGAGAGTTG | ||
| TUB2 | T1 | AACATGCGTGAGATTGTAAGT | Aveskamp et al 2009 |
| Btub4R | CCRGAYTGRCCRAARACRAAGTTGTC |
小孢子链格孢的种类鉴定主要依靠ITS链格孢过敏原基因(Altal)、GAPDH、翻译延伸因子 (TEF1-a)、RNA 聚合酶第二大亚基单位(RPB2)、内聚半乳糖醛酸酶 (endoPG)和OPA10-2 等多基因序列进行系统发育分析 (Woudenberg et al 2015)。
| Gene | Primer | Sequence(5’-3’) | Reference |
| Altal | ALT-F | ATGCAGTTCACCACCATCGC | Hong et al 2005 |
| ALT-R | ACGAGGGTGAYGTAGGCGTC | ||
| TEF1-a | EF1-728F | CATCGAGAAGTTCGAGAAGG | Carboneand Kohn 1999 |
| EF1-986R | TACTTGAAGGAACCCTTACC | ||
| RPB2 | fRPB2-5F | GA(T/C)GA(T/C)(A/C)G(A/T)GATCA(T/C)TT(T/C)GG | Liu et al. 1999。 |
| fRPB2-7cR | CCCAT(A/G)GCTTG(T/C)TT(A/G)CCCAT | ||
| endoPG | PG3 | TACCATGGTTCTTTCCGA | Andrew et al 2009 |
| PG2b | GAGAATTCRCARTCYTGRTT |
黑孢霉属分类鉴定主要采用 ITS、TEFI-a、TUB2 等个组群多基因进行系统发育分析。
真菌分子生物学鉴定部分描述引用自华中农业大学吕闯的硕士论文《广东省葫芦科蔬菜真菌性叶斑病病原鉴定》
- 二、细菌的分子生物学鉴定
细菌的16S rDNA PCR鉴定采用通用引物对27F/1492R进行扩增：
通过通用引物可将细菌鉴定到属水平，要将菌划分到具体是种水平，可选择gyrB和rpoD基因进行种水平的PCR鉴定，引物序列见下表（其中gyrB和rpoD是用P.oryzihabitans的引物作为参考）：
| Gene | Primer | Sequence(5’-3’) | Reference |
| 16s rDNA | 27F | AGAGTTTGATCCTGGCTCAG | Lane, 1991 |
| 1492R | GGTTACCTTGTTACGACTT | ||
| gyrB | gyr AF3 | CCGGCGGTAAGTTCGATGATAACTC | 刘忠玄，2020 |
| gyr AR3 | TGATCGCGGTCAGGCCTTCACGGGC | ||
| rpoD | PsEG30F | ATYGAAATCGCCAARCG | |
| PsEG790R | CGGTTGATKTCCTTGA |
- 三、参考文献
刘忠玄. 花椒细菌性黑腐病病原鉴定、检测及其耐药机理研究[D].东北林业大学,2020.DOI:10.27009/d.cnki.gdblu.2020.000042.
吕闯. 广东省葫芦科蔬菜真菌性叶斑病病原鉴定[D].华中农业大学,2022.DOI:10.27158/d.cnki.ghznu.2022.000295.
Aveskamp MM, V erkley GJM, Gruyter JD, Murace MA, Perelló A, Woudenberg JHC, Crous GPW. DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties. Mycologia, 2009, 101: 363-382.
Andrew M, Peever TL, Pryor BM. An expanded multilocus phylogeny does not resolve morphological species within the small-spored Alternaria species complex. Mycologia, 2009, 101: 95-109.
Carbone I, Kohn LM. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 1999, 91: 553-556.
Carbone, I., & Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91(3), 553–556.
Gardes M, Bruns TD. ITS primers with enhanced specificity for basidiomycetes–application to the identification of mycorrhizae and rusts. Mol Ecol, 1993, 2: 113-118.
Guerber JC, Liu B, Correll JC, Johnston PR. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia, 2003, 95: 872-895.
Hong SG, Cramer RA, Lawrence CB, Pryor BM. Alt a 1 allergen homologs from Alternaria and related taxa: analysis of phylogenetic content and secondary structure. Fungal Genetics & Biology, 2005, 42: 119-129.
Liu Y J and others, Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit., Molecular Biology and Evolution, Volume 16, Issue 12, 1 December 1999, Pages 1799–1808.
