引物设计参考引物
Primers
HP105F2 (5‘-GCTATTGCCTTGCAACAAATCCCCGC-3‘）
HP105R2 (5‘-ATACTTAGGCGGGCATAGCGATG-3‘) were used to PCR-amplify a1752 bp
https://www.uniprot.org/uniprot/O24931
Gene disruption in the mutant (designated HPKY08) was confirmed by PCR using the primer pairs HP105F2
and HP105R2, and
HP02F (5‘-CATACGAAAAAAGCCGCCC-3‘)
and
HP02R (5‘-CTTGTCTGTGGGGATTGAG-3‘), which yielded a
PCR product of increased size. The mutation was also confirmed by
Southern blot analysis with a probe containing the luxS gene. The
mutant HPKY08 was used in all subsequent experiments.
Gene disruption in the mutant (designated
HPKY08) was confirmed by PCR using the primer pairs HP105F2
and HP105R2, and
HP02F (59-CATACGAAAAAAGCCGCCC-39)
and
HP02R (59-CTTGTCTGTGGGGATTGAG-39), which yielded a
PCR product of increased size. The mutation was also confirmed by
Southern blot analysis with a probe containing the luxS gene. The
mutant HPKY08 was used in all subsequent experiments.
Quantitative analysis was performed using the SYBR Green method.
The generation of quantitative data was based on the different PCR
kinetics of samples with various levels of target-gene expression.
cDNA was amplified using PCR primers for H. pylori 16S rRNA,
16S2-F (59-CGCTAAGAGATCAGCCTATGTCC-39) and
16SB2-R(59-CCGTGTCTCAGTTCCAGTGTGT-39). Amplification of the
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene on cDNA
derived from Mongolian gerbils using the primers
G3PDH-F(59-ACCACAGTCCATGCCATCAC-39) and
G3PDH-R (59-TCCACCACCCTGTTGCTGTA-39) (RT-PCR Primer Set, TOYOBO) was used
as a control and for standardization of target gene transcriptional
activity data. For each primer set, PCR was performed in triplicate.
Quantitative data were calculated from a standard curve generated by
amplifying serial dilutions of a known quantity of amplicon. For this
approach, the specificity of the PCR product was confirmed by
dissociation curve analysis (7500 quantification program, Applied
Biosystems)
As shown by the qRT-PCR results (Fig.5b) at 12weeks after
inoculation with HPKY08 (luxS 2), considerable amounts of
mRNA for H. pylori 16S rRNA were detected in the mucus of
gerbil stomach, but isolation of H. pylori from gastric mucus
was negative. We performed an animal experiment using the
luxS-complemented strain HPKY21 in the Mongolian gerbil
model. In the qRT-PCR assay, the number of colonizing H.
pylori luxS-complemented HPKY21 strain cells was more
than that of the luxS 2mutant strain. In addition, there was no significant difference in the number of colonizing H. pylori between TK1402- and HPKY21-infected.
PS：文章里的luxS-cat是什么？
https://aem.asm.org/content/72/10/6615#ref-9