真菌检测鉴定通用引物-Fungal Primers

ITS1: 5’-CCGTAGGTGAACCTGCGG-3’

ITS4:5’-TCCTCCGCTTATTGATATGC-3’ Tm 55℃

NS17: CATGTCTAAGTTTAAGCAA

NS3: GCAAGTCTGGTGCCAGCAGCC

NS4: CTTCCGTCAATTCCTTTAAG

NS22: AATTAAGCAGACAAATCACT

NS24: AAACCTTgTTACgACTTTTA

LR0R: 5’-GTACCCGCTGAACTTAAGC-3’

LR3: 5’-CCGTGTTTCAAGACGGG

LR3R: 5’-GTCTTGAAACACGGACC (complementary to RLR3R: GGTCCGTGTTTCAAGAC)

LR5: 5’-TTAAAAAGCTCGTAGTTGAAC-3’

LR7: 5’-TACTACCACCAAGATCT

LR12: 5’-GACTTAGAGGCGTTCAG

Lr0R/LR5: Tm 50-52℃

NL1: 5’-GCATATCAATAAGCGGAGGAAAAG

NL1: 5′-TGCGTTGATTACGTCCCTGC (also called V9: TGCGTTGATTACGTCCCTGC) NL1: 5’-TGCTGGAGCCATGGATC-3

NL2: 5’-CTCTCTTTTCAAAGTTCTTTTCATCT

NL2: 5’-AACGGCTTCGACAACAGC-3

NL2: 5’-CTTGTTCGCTATCGGTCTC (also NL2A: 5′-CTTGTTCGCTATCGGTCTC)

NL2: 5’-TACTTGTTCGCTATCGGTCT-3'

NL3: 5’-GAGACCGATAGCGAACAAG (also NL3A: 5’-GAGACCGATAGCGAACAAG)

NL3: 5’-AGACCGATAGCGAACAAGTA

NL3: 5’

NL4: 5’-GGTCCGTGTTTCAAGACGG （similar to RLR3R：5′-GGTCCGTGTTTCAAGAC) NL4: 5’-TAGATACATGGCGCAGTC-3

Conserved primer sequences for PCR amplification and sequencing from nuclear ribosomal RNA (/fungi/mycolab/primers.htm)

Vilgalys lab, Duke University

Over the years, our lab has compiled a useful list of conserved primer sequences useful for amplification and sequencing of nuclear rDNA from most major groups of fungi (primarily Eumycota), as well as other eukaryotes. All of these primers were identified and tested by our own lab based on consensus between the published large and small subunit RNA sequences from fungi, plants and other eukaryotes; sources of other useful primer sequences from published literature are also indicated. Together, these primers span most of the nuclear rDNA coding region (see figures), permitting amplification of any desired region. Standard symbols are used for the four primary nucleotides; variable positions are indicated as follows: P=A,G / Q=C,T / R=A,T /

V=A,C / W=G,T. Primers ending with "R" represent the coding strand (same as RNA). All other primers are complementary to the coding strand. This information is provided freely and may be passed on to anyone who wants to use it.

The nuclear-encoded ribosomal RNA genes (rDNA) of fungi exist as a multiple-copy gene family comprised of highly similar DNA sequences (typically from 8-12 kb each) arranged in a

head-to-toe manner. Each repeat unit has coding regions for one major transcript (containing the primary rRNAs for a single ribosome), punctuated by one or more intergenic spacer (IGS) regions. In some groups (mostly basidiomcyetes and some ascomycetous yeasts), each repeat also has a separately transcribed coding region for 5S RNA whose position and direction of transcription may vay among groups. Several restriction sites for EcoRI and BglII are conserved in the rDNA of fungi. Nearly all basidiomycetes we've studied share an EcoRI site within the 5.8S RNA gene along with a BglII site halfway into the LSU RNA sequence. Primers 5.8SR and LR7 include these restriction sites, which makes them convenient for cloning.

For those who aren't familiar with rDNA and fungal systematics, several excellent reviews are available on fungi (Hibbett, 1992) and generally for eukaryotes (Hillis and Dixon, 1991). See Gerbi (1986) for a general introduction to the molecular biology and evolution of rDNA in other eukaryotes. Another useful source of primer information may be found in Gargas & Depriest (1996) and at the Tom Bruns lab web site /boletus/boletus.html.