Single nucleotide polymorphisms and community-associated methicillin-resistant
The present invention is based on the discovery of polymorphisms (SNPs) in the penicillin binding protein (pbp3) gene in Staphylococcus aureus . The presence of G88A and/or G2047A SNPs provides an accurate, reliable biomarker for the presence of Methicillin Resistant Staphylococcus aureus (MRSA), specifically the community-associated MRSA (CA-MRSA). The present invention provides reagents used for detecting the SNPs as well as methods of identifying and using these variants to screen subjects for presence of CA-MRSA. The methods involve isolating a biological sample from a mammal (preferably a human) and testing for the presence of a SNP in the pbp3 gene which is associated with CA-MRSA.
1. A method of determining the presence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in a human, comprising the steps of:
(a) obtaining a biological sample from a human suspected of having a CA-MRSA infection, wherein said biological sample is fluid or tissue;
(b) isolating genomic material from said biological sample;
(c) performing a polymerase chain reaction (PCR) targeted against the penicillin binding protein 3 gene (SEQ ID NO: 39) present in said isolated genomic material to generate an amplicon containing the SNP G88A by using a forward primer consisting of SEQ ID NO: 1 and a reverse primer consisting of SEQ ID NO: 2;
(d) performing a sequencing reaction on said generated amplicon by using a sequencing primer consisting of SEQ ID NO:1;
(e) detecting the presence of adenine (A) at nucleotide position 88 (SNP G88A) in said penicillin binding protein 3 gene in said amplicon; and
(f) determining the presence of CA-MRSA in said human by said presence of said adenine at said nucleotide position 88 (SNP G88A) in said amplicon.
2. The method of claim 1 , wherein said biological sample is whole blood, plasma, urine, or exudate from an infected site.
3. The method of claim 1 , wherein said isolating step is performed using guanidinium hydrochloride.
4. A method of determining the presence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in a human, comprising the steps of:
(a) obtaining a biological sample from a human suspected of having a CA-MRSA infection, wherein said biological sample is fluid or tissue;
(b) isolating genomic material from said biological sample;
(c) performing a polymerase chain reaction (PCR) targeted against the penicillin binding protein 3 gene (SEQ ID NO: 39) present in said isolated genomic material to generate an amplicon containing the SNP G88A by using a forward primer consisting of SEQ ID NO: 3 and a reverse primer consisting of SEQ ID NO: 4;
(d) performing a sequencing reaction on said generated amplicon by using a sequencing primer consisting of SEQ ID NO:3;
(e) detecting the presence of adenine (A) at nucleotide position 88 (SNP G88A) in said penicillin binding protein 3 gene in said amplicon; and
(f) determining the presence of CA-MRSA in said human by said presence of said adenine at said nucleotide position 88 (SNP G88A) in said amplicon.
5. A method of determining the presence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in a human, comprising the steps of:
(a) obtaining a biological sample from a human suspected of having a CA-MRSA infection, wherein said biological sample is fluid or tissue;
(b) isolating genomic material from said biological sample;
(c) performing a polymerase chain reaction (PCR) targeted against the penicillin binding protein 3 gene (SEQ ID NO: 39) present in said isolated genomic material to generate an amplicon containing the SNP G88A by using a forward primer consisting of SEQ ID NO: 5 and a reverse primer consisting of SEQ ID NO: 6;
(d) performing a sequencing reaction on said generated amplicon by using a sequencing primer consisting of SEQ ID NO: 5;
(e) detecting the presence of adenine (A) at nucleotide position 88 (SNP G88A) in said penicillin binding protein 3 gene in said amplicon; and
(f) determining the presence of CA-MRSA in said human by said presence of said adenine at said nucleotide position 88 (SNP G88A) in said amplicon.
6. A method of determining the presence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in a human, comprising the steps of:
(a) obtaining a biological sample from a human suspected of having a CA-MRSA infection, wherein said biological sample is fluid or tissue;
(b) isolating genomic material from said biological sample;
(c) performing a polymerase chain reaction (PCR) targeted against the penicillin binding protein 3 gene (SEQ ID NO: 39) present in said isolated genomic material to generate an amplicon containing the SNP G88A by using a forward primer consisting of SEQ ID NO: 7 and a reverse primer consisting of SEQ ID NO: 8;
(d) performing a sequencing reaction on said generated amplicon by using a sequencing primer consisting of SEQ ID NO: 7;
(e) detecting the presence of adenine (A) at nucleotide position 88 (SNP G88A) in said penicillin binding protein 3 gene in said generated amplicon; and
(f) determining the presence of CA-MRSA in said human by said presence of said adenine at said nucleotide position 88 (SNP G88A) in said generated amplicon.
7. A method of determining the presence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in a human, comprising the steps of:
(a) obtaining a biological sample from a human suspected of having CA-MRSA infection, wherein said biological sample is fluid or tissue;
(b) isolating genomic material from said biological sample;
(c) performing an allele-specific PCR targeted against penicillin binding protein 3 gene (SEQ ID NO: 39) on said isolated genomic material to obtain an amplicon containing the SNP G88A by using a forward primer selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 and a reverse primer selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; and
(d) determining the presence of CA-MRSA in said human as indicated by generation of said amplicon.
8. The method of claim 7 , wherein said forward primer consists of SEQ ID NO: 26 and said reverse primer consists of SEQ ID NO: 36.
9. The method of claim 4 , wherein said biological sample is whole blood, plasma, urine, or exudate from an infected site.
10. The method of claim 5 , wherein said biological sample is whole blood, plasma, urine, or exudate from an infected site.
11. The method of claim 6 , wherein said biological sample is whole blood, plasma, urine, or exudate from an infected site.
12. The method of claim 7 , wherein said biological sample is whole blood, plasma, urine, or exudate from an infected site.
13. The method of claim 4 , wherein said isolating step is performed using guanidinium hydrochloride.
14. The method of claim 5 , wherein said isolating step is performed using guanidinium hydrochloride.
15. The method of claim 6 , wherein said isolating step is performed using guanidinium hydrochloride.
16. The method of claim 7 , wherein said isolating step is performed using guanidinium hydrochloride.