Diagnostic test for bacterial pathogens using internal control bacterial strain
The invention relates to a method for detecting the presence or absence of a bacterial pathogen in a biological sample obtained from a human or animal subject, using an internal control. In particular, the invention relates to a method for detecting the presence or absence of Streptococcus equi in an equine sample using a control bacterial strain as internal control for DNA extraction and PCR. The invention also relates to host cells (such as bacterial cells) and nucleic acids for use as internal standard in said method in addition to diagnostic kits comprising said host cells and nucleic acids.
1. A method for detecting the presence or absence of Streptococcus equi in a biological sample, the method comprising the steps of:
(a) adding a control bacterial strain to the biological sample, wherein the sample is obtained from an animal subject, and wherein the genome of the control bacterial strain contains a target sequence region comprising the nucleotide sequence of: 5′-CCGTGTATTACGCATGCGGGTAGATTATGTAGGTAGAGACATCCAGGTCAAGTTCTCGAC CTTCTCGTGGGAGGTGAACCAG-3′ (SEQ ID NO: 1);
(b) assessing the sample for the presence or absence of the Streptococcus equi SEQ 2190 and/or eqbE gene, wherein detection of the Streptococcus equi SEQ 2190 and/or eqbE gene is indicative of a positive identification for Streptococcus equi in the biological sample; and
(c) assessing the sample for the presence of the target sequence region within the control bacterial strain, wherein detection of the target sequence region within the control bacterial strain is indicative of an accurate result for step (b).
2. The method according to claim 1 , wherein the control bacterial strain is a Streptococcus zooepidemicus strain.
3. The method according to claim 2 , wherein the Streptococcus zooepidemicus strain is Streptococcus zooepidemicus H70.
4. The method according to claim 1 , wherein the sample is obtained from an equine.
5. The method according to claim 1 , wherein the biological sample comprises a nasal swab or wash, pus from an abscess or lavage of the guttural pouch.
6. The method according to claim 1 , wherein the assessing step (b) comprises detecting the Streptococcus equi (eqbE) gene.
7. The method according to claim 6 , wherein detecting the eqbE gene comprises using the primers and probes of SEQ ID NOS: 16, 17 and 20.
8. The method according to claim 1 , wherein the assessing step (b) comprises detecting the Streptococcus equi eqbE and SEQ 2190 genes.
9. The method according to claim 1 , wherein the assessing step (b) comprises detecting the SEQ 2190 gene.
10. The method according to claim 9 , wherein detecting the SEQ 2190 gene comprises using the primers and probes of SEQ ID NOS: 18, 19 and 21.
11. The method according to claim 1 , wherein the sample is obtained from a horse, a donkey or a mule.