Molecular Detection of Streptococcus pyogenes by Strand Invasion Based Amplification Assay

Abstract

Introduction: Streptococcus pyogenes (group A Streptococcus, GAS) is responsible for a variety of highly communicable infections, accounting for 5-15 and 20-30% of sore throat hospital visits in adults and children, respectively. Prompt diagnosis of GAS can improve the quality of patient care and minimize the unnecessary use of antibiotics.

Objective: Our objective was to develop an alternative nucleic acid amplification method for the diagnosis of GAS.

Method: We developed and evaluated a strand invasion based amplification (SIBA) assay to rapidly and specifically detect GAS. The performance of the developed GAS SIBA assay was compared with an established GAS polymerase chain reaction (PCR) assay.

Results: The GAS SIBA assay detected small amounts (ten copies) of S. pyogenes DNA within 13 min. The rapid detection time was achieved in part by optimization of magnesium concentration and reaction temperature. The sensitivity and specificity of the GAS SIBA assay for detection of S. pyogenes from clinical specimens were both 100%, and clinical specimens were detected within ~ 8 min of starting the reaction.

Conclusion: Because the GAS SIBA assay is performed at low and constant temperature, it can be used both in centralized laboratories and for point-of-care testing. Furthermore, given its short detection time and strong analytical performance, the GAS SIBA assay could help to improve patient care and minimize unnecessary prescription of antibiotics.

Fig. 1

DNA amplification by strand invasion based amplification method. 1. The SIBA reaction requires two target-specific primers and IO. Gp32 binds to all single-stranded DNA. 2. The recombinase protein, UvsX, coats the IO, displacing the bound Gp32. The primers are too short to act as substrates for UvsX. 3. The recombinase-coated IO invades the complementary region of the target duplex. The invasion process facilitates the separation of the target duplex, enabling target-specific primers to bind the target. 4. The strand displacement polymerase extends the dissociated target duplex from the primers. 5. This event leads to the production of two copies of the target duplex. Recombinase-mediated target duplex separation and polymerase-mediated extension are the basis for exponential amplification. Image and description were modified from Eboigbodin et al. [13]. IO invasion oligonucleotide, SIBA strand invasion based amplification

Fig. 2

Optimization of GAS SIBA reaction conditions: a magnesium acetate; b reaction temperature. GAS group A Streptococcus, SIBA strand invasion based amplification

Fig. 3

Sensitivity of SIBA and PCR for the detection of Streptococcus pyogenes. a SIBA detection of S. pyogenes ATCC 19615 DNA. b SIBA detection of S. pyogenes NCTC 9994 DNA. c PCR detection of S. pyogenes ATCC 19615 DNA. d PCR detection of S. pyogenes NCTC 9994 DNA. Ct cycle threshold, NTC no template control, PCR polymerase chain reaction, SIBA strand invasion based amplification

Fig. 4

Rapid detection of GAS. a Distribution of time required to detect GAS-positive throat specimens by SIBA and PCR assays. b Comparison of detection times of the GAS SIBA and GAS PCR assays. *GAS SIBA and GAS PCR assay detection times are reported in minutes and Ct, respectively. Ct cycle threshold, GAS group A Streptococcus, PCR polymerase chain reaction, SIBA strand invasion based amplification