Introduction

Streptococcus pneumoniae is a non-flagellated, Gram-positive bacterium that causes invasive pneumococcal disease (IPD) in humans1,2. It is estimated that over 1.6 million people, including more than 800,000 children under the age of 5, die each year from pneumococcal infections3,4. S. pneumoniae can cause otitis media, bloodstream infections, spontaneous peritonitis and meningitis5. Pneumococcal meningitis is a form of pneumococcal disease that causes significant morbidity and mortality in children6.

In the Streptococcus genus, distinguishing between colonies of S. pneumoniae and other Streptococcus isolates based on morphology and immunogenicity can be challenging due to their striking similarities7,8,9. Therefore, identifying S. pneumoniae and other closely related Streptococcus isolates presents a significant challenge for clinical microbiology laboratories. Rapid and accurate diagnosis and identification of S. pneumoniae are crucial for the treatment and prevention of IPD and its complications10,11,12. Although the culture-based method is considered the gold standard for diagnosing and identifying S. pneumoniae, it typically takes 3–5 days and may result in missed opportunities for timely treatment. Furthermore, this method has limited diagnostic sensitivity, particularly in cases where prior antibiotic treatment has been administered. As a result, it is likely that many cases have been misdiagnosed or undiagnosed. The ELISA method can be used to detect specific antibodies against S. pneumoniae13. However, due to the high positivity rate of antibodies in the epidemic area, this method has limited diagnostic value. Additionally, S. pneumoniae antigens have common cross-reactivity with other Streptococcus isolates. Therefore, the specificity of S. pneumoniae detection by ELISA method is not high. The PCR-based assays frequently target the lytA gene, which encodes pneumococcal virulence factors, for identifying S. pneumoniae14,15. However, they currently have drawbacks such as cumbersome operational procedures, susceptibility to pollution, time-consuming processes, and require expensive equipment as well as skilled personnel. Therefore, there is an urgent need to investigate a novel method that can rapidly and accurately differentiate S. pneumoniae from other non-S. pneumoniae bacterial species.

Multienzyme isothermal rapid amplification (MIRA) is an emerging technique for isothermal amplification that eliminates the need for skilled personnel and complicated, time-consuming procedures16,17,18. The technique is dependent on three key enzymes, namely recombinase, single-stranded DNA binding protein (SSB), and DNA polymerase19. The amplification process is initiated by a primer-recombinase complex, which can invade the DNA double strand at homologous sequences of the primer and initiate amplification. Subsequently, the single-stranded binding protein (SSB) stabilizes the reaction, allowing for polymerase extension. This entire process can be completed within 8–10 min at a temperature of 37–42 °C, rendering it an optimal technique for point-of-care testing. Compared to existing isothermal amplification technologies such as LAMP, RPA, SDA, HDA, and NASBA, MIRA technology has lower sample requirements, freeing it from the limitations of imported raw materials20,21,22. Additionally, it can be combined with other detection techniques, further expanding the applicability of this technology. In recent years, MIRA technology has garnered widespread attention due to its high sensitivity, specificity, short detection time, isothermal reaction at room temperature, diverse detection applications, lyophilized reagents for easy transportation, and stable enzyme activity. Thus, the current study aims to develop a rapid, simple, and reliable real-time MIRA assay based on optimization of MIRA primer combinations and reaction conditions for rapid detection of S. pneumoniae. A primer–probe set targeting the S. pneumoniae lytA gene will be designed, followed by optimization of parameters for the MIRA assay. At the same time, we will validate the real-time MIRA assay for detecting S. pneumoniae using 79 clinical isolates identified by VITEK MS.

Methods

Bacterial strains and genomic DNA preparation

A total of 79 clinical isolates were collected from Daping Hospital, including thirty S. pneumoniae, seven Streptococcus constellatus, six Enterococcus faecium, four Streptococcus salivarius, four Enterococcus faecalis, three Streptococcus anginosus, three Escherichia coli, three Klebsiella pneumoniae, three Stenotrophomonas maltophilia, two Streptococcus epidermidis, two Acinetobacter baumannii, two Pseudomonas aeruginosa, two Enterococcus avium and one each of Proteus penneri, Acinetobacter lwoffii, Staphylococcus aureus, Citrobacter freudii, Proteus hauseri, Burkholderia cenocepacia, Candida albicans, and Enterobacter aerogenes isolate. The identification of all these isolates used in this study was confirmed at the species level by VITEK MS (Figure S1). The genomic DNA of all these isolates was extracted using the TIANGEN genomic DNA isolation kit (TIANGEN, Beijing, China) following the manufacturer's instructions. DNA quantification was performed using a NanoDrop spectrophotometer (Bio-Rad, USA). This instrument allows for rapid and accurate measurement of nucleic acid concentrations based on their absorbance at specific wavelengths. Briefly, DNA samples were prepared according to the manufacturer's instructions and pipetted onto the sample pedestal of the NanoDrop spectrophotometer. The instrument was then calibrated, and the absorbance of the samples was measured at a wavelength suitable for DNA quantification (typically around 260 nm). The NanoDrop software automatically calculated the DNA concentration based on the absorbance readings and provided the results in units such as pg/µL or pg/reaction. S. pneumoniae ATCC49619 was selected as the standard strain. Culturing and analysis of all these strains were performed in a biosafety level two mycobacteriology laboratory at Daping Hospital, following biosafety level two precautions23.

Primer and probe design

The real-time MIRA assay was designed using primers based on the S. pneumoniae-specific lytA gene (GenBank accession no. AF467249.1), which has previously been utilized in PCR-based assays for specific detection of S. pneumoniae24,25. The primers and probes were manually designed within the conserved regions of the lytA gene, following the principles of real-time MIRA primer and probe design. Primer-BLAST from NCBI was employed to validate their specificity, while online OligoEvaluator software (http://www.oligoevaluator.com) was utilized for analyzing potential primer dimers and hairpins. All primers and probes (listed in Table 1) were synthesized and purified by BGI Biotechnology Corporation (Beijing, China) using high-performance liquid chromatography (HPLC).

Table 1 Sequences of primer and probe.
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Real-time MIRA assay

The real-time MIRA reaction was achieved using exo MIRA kits (Amp-Future Biotech Co., Ltd., Weifang, China). A real-time MIRA reaction consisted of 2 µL of DNA template, 29.5 µL of reaction buffer, 11.2 µL of water, 2.1 µL of forward primer (10 µM), 2.1 µL of reverse primer (10 µM), 0.6 µL of probe (10 µM), and 2.5 µL of 280 mM magnesium. The reaction mixture was briefly vortexed and centrifuged briefy. Then, the reactions were immediately transferred into the CFX96 real-time PCR system (Bio-Rad, USA) with a setting of one cycle per 30 s for 20 min (40 cycles) at a constant temperature of 39 °C to monitor fluorescence signals in real-time. A negative control with nuclease-free water was included in each run.

Analytical specificity and sensitivity of the real-time MIRA assay

In order to assess the specificity of the real-time MIRA assay, we conducted cross-reactivity tests on S. pneumoniae, S. aureus, S. saprophytics, E. faecalis, S. mitis, S. dysgalactiae, and S. agalactiae for possible interference with the assay performance. The analytical sensitivity of the real-time MIRA assay was assessed by using known amounts of genomic DNA from S. pneumoniae ATCC49619, and the concentration of target DNA sequence was also evaluated. For this purpose, tenfold serial dilutions of the genomic DNA ranging from 1.7 × 105 to 1.7 × 10–1 pg pre reaction was used as template. The assay was performed in triplicate and consistent results were obtained.

Real-time PCR assay

For comparison, the diluted S. pneumoniae ATCC49619 DNA samples were tested in parallel using an established real-time PCR protocol. The protocol involved heating at 95 °C for 5 min followed by 40 cycles of heating at 95 °C for 10 s and then cooling to 60 °C for 40 s. The detection kit used contained specific primers and probes (listed in Table 1), Premix Ex Taq (Probe qPCR), and double-distilled water. Each reaction mixture consisted of a total volume of 20 µL PCR mix and included an additional volume of 2 µL DNA sample. The threshold cycle (Ct value) < 35 was determined as positive sample.

Evaluation of the real-time MIRA assay using clinical isolates

To evaluate the performance of the real-time MIRA assay for identifying of S. pneumoniae, 79 clinical isolates of both S. pneumoniae and other non-S. pneumoniae bacterial species were tested, and its performance was compared to that of a real-time PCR assay for identifying S. pneumoniae. This experiment was conducted in duplicate.

Ethics approval

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of the Daping Hospital (approval No. 2021223). Written informed consent was obtained from all patients.

Results

Optimal condition of real-time MIRA

The selection of appropriate primers and probes is crucial for conducting real-time MIRA in pathogen detection. In this study, we screened eight pairs of primers and one probe targeting the conserved region of S. pneumoniae lytA gene to obtain the optimal combination. Amplification curves showed that all combinations produced positive signals (Figure S2). However, primer pairs lytA-F1/R2 performed better in terms of amplification time and signal intensity compared to other primers. Therefore, lytA-F1/R2 were selected for further analysis.

Specificity and sensitivity of MIRA assay

Specific amplification was observed with S. pneumoniae ATCC49619 and one clinical S. pneumoniae strain, and there were no cross-detections of other pathogens tested (Fig. 1). To determine the minimum detection limit of the MIRA method, a range of genome copy dilutions ranging from 1.7 × 105 to 1.7 × 10–1 pg were utilized as templates for amplification in each reaction using two distinct methods (real-time MIRA and real-time PCR). The amplification curve depicted in Fig. 2 demonstrates that the lowest concentration of S. pneumoniae ATCC49619 DNA for real-time MIRA was significantly better than that of real-time PCR, reaching 1.7 × 100 pg per reaction. Three independent reactions were repeated, and similar results were observed, demonstrating the good repeatability of this assay.

Figure 1
figure 1

Specificity of the real-time MIRA assay for lytA gene of S. pneumoniae detection. Partial results display that only the S. pneumoniae samples (NO. 1 and 2) produced amplification signals, whereas the other pathogen samples (NO. 3–9) did not produce any amplification signals. This experiment was repeated three times with consistent result.

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Figure 2
figure 2

Sensitivity of the real-time MIRA (a) and real-time PCR (b). The analytical sensitivity of the real-time MIRA assay was tested based on the quantity of genomic DNA from S. pneumoniae ATCC49619. Serially diluted gDNA samples (ranging from 1.7 × 105 to 1.7 × 10–1 pg per reaction) were analyzed using real-time MIRA at 39 °C with a setting of one cycle per 30 s for 20 min (40 cycles) for real-time monitoring of fluorescence signals (a) and real-time PCR at 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 10 s and annealing/extension at 60 °C for 30 s (b). This experiment was repeated three times with consistent results.

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Validation of real-time MIRA assay on clinical isolates

A total of 79 clinical isolates were evaluated using the real-time MIRA assay and compared to results obtained from the reference method, real-time PCR26,27. As shown in Table 2, all DNA samples extracted from S. pneumoniae displayed positive signals, while no signals were detected for DNA samples from other non-S. pneumoniae bacterial species. The real-time MIRA demonstrated a desired reliability of 100% (30/30), indicating promising potential for clinical sample applications. Compared to the real-time PCR assay, the lytA gene-based real-time MIRA showed both sensitivity and specificity at 100%.

Table 2 Characteristics of the 79 clinical isolates used for validation of the designed real-time MIRA assay for detecting S. pneumoniae.
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Discussion

Compared with other related technologies such as LAMP, RPA, SDA, HDA, and NASBAl, the real-time MIRA assay offers several advantages, including high sensitivity, rapid detection time, convenient operation, and less requirement for specialized equipment28,29. Additionally, the amplification process is completed under an isothermal condition at 39 °C within 20 min in a single run without the need to open the lid during the process. Therefore, real-time MIRA amplification results are obtained superiorly compared to other rapid molecular detection methods. In this study, we report on the development and validation of a real-time MIRA assay for rapid detection of S. pneumoniae in clinical isolates. The entire process from MIRA amplification to fluorescence signal detection takes about 20 min or less.

When comparing different target genes of S. pneumoniae, it was found that lytA-based PCR reaction was the most specific and could effectively distinguish S. pneumoniae from atypical Streptococcus30,31. In the present study, six sets of real-time MIRA primers and probes were separately designed based on the lytA gene. This was done after testing a series of real-time MIRA primers and selecting lytA-F1/R2 for use in the real-time MIRA assay. The real-time MIRA assay exhibited high species specificity, enabling it to detect all S. pneumoniae isolates. Additionally, there was no cross-reactivity with other non-S. pneumoniae bacterial species under the experimental conditions used, indicating that real-time MIRA has good specificity. Further studies should focus on verifying the potential cross-reactivity of DNA using the real-time MIRA assay with other non-S. pneumoniae bacterial species to confirm the high specificity (100%) of the assay for detecting S. pneumoniae. Therefore, the real-time MIRA assay is highly reliable in terms of specificity. The sensitivity of the real-time MIRA assay was determined by serially diluting genomic DNA from S. pneumoniae ATCC49619. Our results showed that the established real-time MIRA assay has a sensitivity up to 1.7 × 100 pg, which is better than that of real-time PCR. Next, we used the real-time MIRA assay to detect 79 clinical isolates, and the results showed that 30 clinical isolates were retrospectively confirmed as S. pneumoniae by the real-time MIRA assay, while 49 clinical isolates from non-S. pneumoniae species tested negative for lytA gene detection. The positive detection rate of the real-time MIRA assay was 100%, indicating that it is highly practical with no false positive results.

However, the limitation of this study, however, is that the validation strains were limited. To ensure the accuracy and reliability of the identification results, we need to further expand the number and types of validation strains in order to obtain better identification results. Further studies are required to test the specificity and sensitivity of the present method on clinical blood culture samples that tested positive for Gram-negative bacteria in Gram stain.

In conclusion, this study demonstrates that the designed real-time MIRA assay exhibits high specificity and sensitivity, providing a promising, rapid, simple, and reliable method for detecting S. pneumoniae suitable for use in under-equipped diagnostic laboratories.