Detection of Staphylococcus aureus by a Sensitive Immuno-PCR Assay

Abstract

Staphylococcus aureus causes a wide variety of diseases in humans, the clinical courses of which range from boils and furuncles to more serious diseases such as septicemia and pneumonia (1 ). Although a significant cause of community-acquired infection, most life-threatening cases of S. aureus disease are hospital-acquired and are associated, in many cases, with indwelling vascular devices or catheters (2 ). The standard method of diagnosing S. aureus is to culture an isolate from a blood agar plate and then use a latex test to identify S. aureus.A specific product of S. aureus is protein A (3 ). Protein A is a cell-wall constituent of S. aureus and is mainly covalently linked to the peptidoglycan structure (4 ); however, ϳ8 -30% of the protein is secreted into the medium during the exponential growth phase (5 ). This property has been used to develop a latex agglutination test and an ELISA to identify and detect S. aureus. We describe here a sensitive immuno-PCR assay to detect S. aureus protein A. After optimization of the reaction conditions and the use of flexible plates with 96 V-bottomed wells, which were compatible with both the ELISA washer and the thermal cycler for PCR, we automated both detection methods.Anti-protein A antisera were created by immunizing rabbits with protein A purified from preparative polyacrylamide gels. The IgG fraction of the antisera was purified by DEAE ion-exchange chromatography (6 ). The protocol for biotinylating the antibody is described elsewhere (7 ). The avidin-biotinylated DNA complex was prepared fresh before use by mixing avidin and the biotinylated DNA at a molar ratio of 1:4. Avidin (2 g/L) was first diluted with bovine serum albumin (BSA) diluent to 2 g/L, and then 10 L was mixed with 2 L of the biotinylated DNA (120 mg/L). The mixture was incubated at room temperature for 10 min and then further diluted with BSA diluent before use.The protocol for immuno-PCR was a modified version of the modified protocol of Chang and Huang (8 ) (Fig. 1A). Briefly, microtiter plates were coated with 50 L/ well of anti-protein A IgG (10 mg/L in phosphate-buffered saline) for 1 h at 37°C, washed five times, and blocked with 75 L of the BSA diluent for 1 h at 37°C. The plates were washed five times, and 50 L of 10-fold serial diluted antigen (protein A; 10 Ϫ9 -10 Ϫ18 g/mL) was added to each well. After incubation for 1 h at 37°C, the plates were washed five times, and 50 L of the biotinylated anti-protein A (1 g/L) was added to each well. The plates were incubated at 37°C for 1 h and then washed 12 times; 50 L of the avidin-biotinylated DNA complex (1:10 000 dilution) was then added to each well. The plates were again incubated at 37°C for 1 h and washed 12 times before the PCR step. The negative control was performed in the same way, except that the BSA diluent was substituted for the antigen solution. The buffer used throughout for plate washing was phosphate-buffered saline containing 0.5 mL/L Tween 20, whereas the BSA diluent was used for the dilution of antigen, biotinylated an...

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles

“…This difference in copy numbers can be several orders of magnitudes, and higher target numbers suggest a lower limit of detection. Immuno-PCR is a technology that can ultrasensitively detect bacterial surface antigen using an antibody chimera with a DNA barcode15. However, conjugating DNA molecules onto a specific site of an antibody without affecting its interaction can be problematic.…”

“…However, ultrasensitive detection with such approaches is limited by the fact that antibodies are proteins and thus cannot be amplified. This limitation was circumvented by the development of a technology called immuno-PCR, in which the antibody is cross-linked with a DNA “barcode” for PCR amplification15. Although this technology is sensitive, the conjugation and purification of antibody-DNA complexes is still a daunting task.…”

Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

“…iPCR, which herein has demonstrated successful detection of spirochetes directly from whole blood and is much less prone to the same contamination issues, as the PCR template is unrelated to B. burgdorferi and human DNA, could effectively be used to make a more rapid diagnosis from B. burgdorferi-infected blood cultures. Future work will focus on improving the limit of iPCR direct detection of spirochetes in blood to achieve a detection sensitivity of 1 to 10 organisms, as has been demonstrated for other microbial pathogens (10,(34)(35)(36). Furthermore, as B. burgdorferi is transiently present in the blood of infected patients, the iPCR method may also be adapted for direct detection of spirochetes in synovial fluid and/or cerebrospinal fluid.…”

Immuno-polymerase chain reaction as a unique molecular tool for detection of infectious agents

“…Specifc examples of these infectious agents that have been analyzed using IPCR are: HIV-1 p24 Ag [23] ; rotavirus VP6 Ag [24] ; Hantaan virus nucleocapsid protein [7] ; norovirus capsid proteins [25] ; Staphylococcus aureus protein A [26] ; and group A Streptococcus bacteria [27] .…”

“…Purified protein A Ag dilutions, as well as patient wound samples, were used with the indirect molecular format 2b ( Figure 2 ), with flexible V-bottomed plates (unknown manufacturer) for both the ELISA and PCR tests [26] . The avidin-biotinylated DNA reporter template was prepared by mixing the 2 molecules (avidin and biotinylated DNA) at a molar ratio of 1:4, added as a 1-step reagent.…”