Computational protocol: Molecular detection of colistin resistance genes (mcr-1 to mcr-5) in human vaginal swabs

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Protocol publication

[…] This study received permission from the patients and was approved by the Institutional Review Board of Subei People’s Hospital. In 2016, vaginal swabs were collected from 134 women attending Subei People’s Hospital of Yangzhou in China for first or second infertility evaluation. Previous study demonstrated that none of vaginal swabs were positive for Neisseria gonorrhoeae and Treponema pallidum, but 18.8% of these swabs were positive for Chlamydia trachomatis and 17.3% of the swabs were positive for Mycoplasma species by PCRs []. All swabs were positive for tetracycline resistance gene tet(M) which is the most effective antibiotic for bacterial sexually transmitted infections [].Collection and DNA extraction of the human vaginal swabs were performed as described before []. Nucleotides of mcr-1, mcr-2 and mcr-3 genes in the samples were amplified with primers described before []. Meanwhile, using the Clustal Multiple Alignment Algorithm, we developed and validated a mcr-4-PCR (forward primer: 5′-AATTGTCGTGGGAAAAGCCGC-3′; reverse primer: 5′-CTGCTGACTGGGCTATTACCGTCAT-3′; amplicon size 1062 bp), and a mcr-5-PCR (forward primer: 5′-GTGAAACAGGTGATCGTGACTTACCG-3′; reverse primer: 5′-CGTGCTTTACACCGATCATGTGCT-3′; amplicon size 271 bp) in this study. The specificity of the primers for mcr-4 and mcr-5 PCRs were verified by BLASTN and DNA sequencing of the obtained PCR products. The sensitivity of the mcr-4-PCR and mcr-5-PCR was determined by amplifying dilutions of synthesized plasmids containing portions of the target mcr-4 and mcr-5 which were linearized with Sac I (Takara Biothechnology, Dalian, China). The quantitative standards were quantified using the PicoGreen DNA fluorescence assay (Molecular Probes, Eugene, OR, USA) for preparation of standards (104, 103, 102, 101, and 100 copies/reaction). The PCR products were confirmed by gel electrophoresis, and DNA sequencing with both PCR primers (BGI, Beijing, China) after purification with QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA).In this study, every 24th samples tested consisted of diethylpyrocarbonate-treated ddH2O, serving as a negative extraction control to confirm the absence of contamination between samples during DNA extraction and carry-over contamination. Additionally, control swabs from pipettes, experimental benches and centrifuges were frequently processed for five mcr-PCRs to verify that no false amplification occurred during this study resulting from carry-over contamination.All reported human mcr sequences, representative mcr sequences from animals, and the mcr sequences from animals at Yangzhou were aligned with the obtained mcr sequences in this study. Based on these alignments, phylogenetic trees were constructed by the neighbor-joining method using the Kimura 2-parameter model with MEGA 6.0. The Bootstrap values were calculated using 500 replicates. The BLASTN was performed to determine new mcr variants by comparing the mcr sequences from this study and those available in GenBank. […]

Pipeline specifications

Software tools BLASTN, MEGA
Application Phylogenetics
Organisms Homo sapiens, Bacteria
Diseases Infertility
Chemicals Colistin, Nucleotides