Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum.
Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63 °C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production.
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Yabing
Y
Duan
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Xiaoke
X
Zhang
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Changyan
C
Ge
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Yong
Y
Wang
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Junhong
J
Cao
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Xiaojing
X
Jia
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Jianxin
J
Wang
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Mingguo
M
Zhou
D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology*; D002219:Carbamates / Q000494:pharmacology*; D004396:Coloring Agents / Q000737:chemistry; D017931:DNA Primers / Q000138:chemical synthesis; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D005659:Fungicides, Industrial / Q000494:pharmacology*; D005670:Fusarium / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology; D017354:Point Mutation; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D014908:Triticum / Q000382:microbiology; D014404:Tubulin / Q000032:analysis* / Q000235:genetics
D001562:Benzimidazoles; D002219:Carbamates; D004396:Coloring Agents; D017931:DNA Primers; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D009282:Naphthalenesulfonates; D020033:Protein Isoforms; D014404:Tubulin; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate; C006698:carbendazim