A multiple method for sensitively detecting 17 highly infectious bacteria and viruses with distinguished melting peaks

Abstract

Background: Multiple fluorescence PCR was used to facilitate the low cost detection of such infectious pathogens. The melting curve combined with the fluorescence PCR method was used to detect more targets. The characteristic melting peaks of the melting curve formed by the melting probe were complementary to the fluorescent probes and more targets could be identified by the different melting peaks in one fluorescence channel. Results: The most optimal conditions were confirmed by optimizing the experimental conditions, such as the concentration of fluorescent probes and the melting probe, which is crucial for forming the characteristic melting peaks. LOD, multiplex performance and sample detection performance were evaluated separately. According to the results, the LOD of all targets was as low as five or ten copies/tube, excepting for Zaire type Ebola virus, whose LOD is 25 copies/tube. The method could simultaneously detected more targets, even up to eight bacteria or nine viruses and the result was accurate. In 173 clinical or real-world samples, the result was in keeping with the expected or clinical results, excepting for one case of the soil sample, and the consistency rate of the detection results reached 99.42%. Significance: We achieved a novel method for multiple detection of up to 17 highly infectious pathogenic microorganisms and performed well in real sample detection, and the results were consistent with the expected or clinical results. We provided a new detection tool for disease prevention, control centers and clinical practice.

Keywords

• Multiplex fluorescent PCR
• Melting curve
• Microbial pathogens
• Highly infectious bacteria
• Highly infectious viruses