A highly sensitive immuno-PCR assay for detecting Group A Streptococcus.

A highly sensitive hybrid assay, based on immuno polymerase chain reaction (immuno-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques, was developed for the detection of pathogenic Group A Streptococcus (Strep A). Cells were disrupted by sonication and then coated onto the walls of Maxisorp microtiter plates. Next, biotinylated anti-Group A monoclonal antibody (mAb) was bound to the antigen and then linked, via a streptavidin (STV) bridge, to biotinylated reporter DNA. After extensive washing, the denatured reporter DNA was transferred to PCR tubes, amplified, electrophoresed, and used as the signal for detection of bacteria. The minimum detection limit of this assay is the equivalent of approximately one one-thousandth of a Streptococcus pyogenes cell, even in the presence of 100,000 Escherichia coli cells. The combination of multiple antigens per cell and PCR amplification provides the extreme sensitivity in this immuno-PCR assay. No cross-reaction was found with other Streptococcus species. We also directly linked the anti-Group A monoclonal antibody to DNA using succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The sensitivity using directly linked antibody-reporter DNA was approximately 10 cells. Because this assay could be adapted for detection of many different bacteria in a variety of sample types, we tested the potential for interference from substances that could be present in clinical, food, and environmental samples. Sonicated meat or human plasma did not inhibit detection; however, extracts of concentrated soil samples were somewhat inhibitory. This highly specific, sensitive, and robust assay could be applied to clinical detection of Group A Streptococcus and serves as a model for other immuno-PCR assays.

[1]  P. Askenase,et al.  Soluble T cell receptors: detection and quantitative assay in fluid phase via ELISA or immuno-PCR. , 1995, Journal of immunological methods.

[2]  R. Fisher,et al.  Universal immuno-PCR for ultra-sensitive target protein detection. , 1993, Nucleic acids research.

[3]  Darrell P. Chandler,et al.  Renewable microcolumns for automated DNA purification and flow-through amplification: from sediment samples through polymerase chain reaction , 2002 .

[4]  R. Joerger,et al.  High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction. , 1995, Nucleic acids research.

[5]  J. Silver,et al.  Nanoliter scale PCR with TaqMan detection. , 1997, Nucleic acids research.

[6]  E. Risseeuw,et al.  Nucleotide sequence corrections of the uidA open reading frame encoding beta-glucuronidase. , 1994, Gene.

[7]  E. Pich,et al.  Rapid induction of tumor necrosis factor alpha in the cerebrospinal fluid after intracerebroventricular injection of lipopolysaccharide revealed by a sensitive capture immuno-PCR assay. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  N. Tsuji,et al.  Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. , 2001, Clinical chemistry.

[9]  D. Fan,et al.  Detection of circulating gastric carcinoma‐associated antigen MG7‐Ag in human sera using an established single determinant immuno‐polymerase chain reaction technique , 2000, Cancer.

[10]  Lai,et al.  Detection of Clostridium botulinum neurotoxin type A using immuno‐PCR , 2001, Letters in applied microbiology.

[11]  D. Kobayashi,et al.  Detection of human serum tumor necrosis factor-alpha in healthy donors, using a highly sensitive immuno-PCR assay. , 1999, Clinical chemistry.

[12]  D. Stallknecht,et al.  Mycoplasmal conjunctivitis in wild songbirds: the spread of a new contagious disease in a mobile host population. , 1997, Emerging infectious diseases.

[13]  R. Joerger,et al.  Analyte detection with DNA-labeled antibodies and polymerase chain reaction. , 1995, Clinical chemistry.

[14]  T. C. Chang,et al.  A modified immuno-polymerase chain reaction for the detection of beta-glucuronidase from Escherichia coli. , 1997, Journal of immunological methods.

[15]  K. Livak,et al.  Real time quantitative PCR. , 1996, Genome research.

[16]  D. Stevens.  Streptococcal toxic-shock syndrome: spectrum of disease, pathogenesis, and new concepts in treatment. , 1995, Emerging infectious diseases.

[17]  D Blohm,et al.  Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates. , 1997, Analytical biochemistry.

[18]  H. Kida,et al.  A rapid and highly sensitive method for diagnosis of equine influenza by antigen detection using immuno-PCR. , 2001, The Japanese journal of veterinary research.

[19]  D Blohm,et al.  Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR. , 1999, Nucleic acids research.

[20]  D. Kobayashi,et al.  A highly sensitive immuno-polymerase chain reaction assay for human angiotensinogen using the identical first and second polyclonal antibodies. , 2000, Clinica chimica acta; international journal of clinical chemistry.

[21]  C R Cantor,et al.  Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. , 1992, Science.

[22]  P. Choudary,et al.  Adsorption of endogenous polyphenols relieves the inhibition by fruit juices and fresh produce of immuno-PCR detection of Escherichia coli O157:H7. , 1999, FEMS immunology and medical microbiology.

[23]  J. Musser,et al.  Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: recent intercontinental spread of a subclone causing episodes of invasive disease , 1995, Infection and immunity.

[24]  J. Grate,et al.  Automated immunomagnetic separation and microarray detection of E. coli O157:H7 from poultry carcass rinse. , 2001, International journal of food microbiology.

[25]  G. Knudsen,et al.  Isolation and purification of bacterial community DNA from environmental samples. , 1997 .