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

• The characteristic melting peaks of the melting curve formed by the melting probe were complementary to the fluorescent probes and the target could be identified by the different melting peak. • The LOD for detecting the pathogens was as low as five or ten copies per reaction, excepting for the Zaire type Ebola virus , which has a sensitivity of 25 copies per reaction. • The method simultaneously detected different numbers of targets, even up to eight bacteria or nine viruses and the results were accurate. • The real-world samples could be exactly detected, and the results of the part samples were consistent with the results of the single qPCR testing. 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. 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%. 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. Multiple fluorescence PCR was used to facilitate the low cost detection of such infectious pathogens by the characteristic melting curve formed by the melting probe (mP) complementary to the fluorescent probes (fP) and two or more targets could be identified by the different melting peaks in one fluorescence channel. The LOD of this method for detecting the pathogens was as low as five or ten copies per reaction, excepting for Zaire type Ebola virus , which has a sensitivity of 25 copies per reaction, and the method simultaneously detected different numbers of targets, even up to eight bacteria or nine viruses.