Detection and typing of bacterial strains
View Patent ↗Methods for the detection and typing of bacterial strains from food products and dietary supplements, environmental samples, in vivo/in vitro samples, and for studying the natural diversity of the species are disclosed. Potential applications also include product development and/or detection and differentiation of new bacterial strains.
1. A method for typing a Lactobacillus species having a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) region, comprising:
a) obtaining a sample comprising said Lactobacillus;
b) amplifying a region of DNA comprising said CRISPR region or a fragment thereof in said sample to create amplified DNA, ; and
c) typing a Lactobacillus species having a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) region.
2. The method of claim 1 comprising the further steps;
c) adding to said amplified DNA at least one restriction enzyme that recognizes one or more sites in said amplified DNA;
d) incubating said restriction enzyme with said amplified DNA for a time sufficient to form restriction fragments;
e) determining the number of said restriction fragments and their size; and,
f) typing said bacterium based on said number and size of said restriction fragments.
3. The method of claim 1 , further comprising the step of sequencing said amplified DNA.
4. A method for typing a Lactobacillus species having a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) region, comprising:
a) obtaining a sample comprising said Lactobacillus;
b) amplifying a region of DNA comprising said CRISPR region or a fragment thereof in said sample to create amplified DNA, the CRISPR region including at least one CRISPR repeat region or CRISPR spacer region or a portion thereof; and
c) typing a Lactobacillus species having a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) region.
5. The method of claim 4 comprising the further steps;
c) adding to said amplified DNA at least one restriction enzyme that recognizes one or more sites in said amplified DNA;
d) incubating said restriction enzyme with said amplified DNA for a time sufficient to form restriction fragments;
e) determining the number of said restriction fragments and their size; and,
f) typing said bacterium based on said number and size of said restriction fragments.
6. The method of claim 4 , further comprising the step of sequencing said amplified DNA.
7. The method of claim 4 , wherein amplifying a region of DNA includes providing a first primer that binds to a repetitive sequence in a CRISPR region.
8. The method of claim 7 , wherein amplifying a region of DNA includes providing a second primer that binds to DNA flanking the CRISPR region.