Screening and monitoring the progression of type 2 diabetes by the molecular identification of human gut flora using FTA as a faecal collection device
Disclosed is a method for detecting and quantifying gut flora derived from human faeces, comprising acquiring a solid support containing human faeces; optionally amplifying nucleic acid from the human faeces; and detecting and quantifying the presence of gut flora of interest. Also disclosed is a method for assessing whether a person has type-2 diabetes as well as a kit for detecting and quantifying gut flora from human faeces, comprising a solid support for collecting human faeces; and primer pairs for amplifying 16S rRNA sequence from bacteria of interest.
1. A method for detecting gut flora derived from human faeces, comprising
1) acquiring a solid support containing human faeces, wherein the solid support comprises cellulose fibre material, wherein the support has a surface impregnated with chemicals including a weak base, a chelating agent, an anionic surfactant and/or a chaotropic agent;
2) amplifying nucleic acid from the human faeces, wherein the amplifying step is performed by multiplexed PCR using primer pairs for 16S rRNA from the flora using lyophilized phi29 DNA polymerase in the presence of cyclodextrin; and
3) detecting and quantifying from the amplified nucleic acid a ratio of an amount of butyrate-producing bacteria to an amount of sulphate reduction/oxidative stress resistance bacteria.
2. The method of claim 1 , wherein the human faeces had been applied to the solid support and dried prior to step 1 .
3. The method of claim 1 , wherein the solid support further comprises a porous polymer, for example porous membrane material such as polyester, polyether sulfone (PES), polyamide (Nylon), polypropylene, polytetrafluoroethylene (PTFE), polycarbonate, cellulose nitrate, cellulose acetate, alginate or aluminium oxide.
4. The method of claim 1 , wherein the chaotropic agent is guanidinium thiocyanate.
5. The method of claim 1 , wherein the amplifying step or the detecting and quantifying step is carried out directly from punches excised from solid support containing the human faeces.
6. The method of claim 1 , further comprising a step of purifying the nucleic acid from the solid support containing the human faeces prior to the amplifying step.
7. The method of claim 1 , wherein detecting and quantifying the 16S rRNA is by next generation sequencing technique.
8. The method of claim 1 , wherein detecting and quantifying the gut flora of interest is by detecting and quantifying a signature nucleic acid sequence for each microorganism of interest using next generation sequencing technique.