Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification

Abstract

Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0 × 10 −17 M (approximately 6.0 × 10 3 copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0 × 10 4 copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the "gold standard" real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens.

Accurate, rapid and highly sensitive detection of African swine fever virus via graphene oxide-based accelerated strand exchange amplification

“…The reaction mixture was kept in narrow thermal cycles between the denaturation temperature (T d ) and the renaturation temperature (T r ) using LineGene Mini S (Bioer, Hangzhou, China). Referring to the T m values of primers, 74 C was the preferred temperature for T d , and the T r would be optimized between 60 and 65 C. 16,17 Therefore, the reaction procedure consisted of 45 cycles performed at 74 C for 1 s and 60-65 C for 1 s, with heating and cooling rates of 5 C s À1 and 4 C s À1 during thermal cycling, respectively. The uorescent signal emanating from the amplication product was scanned for each thermal cycle and plotted over time to monitor the amplication in real time.…”

“…As the amplication reaction progresses, short amplicons were denatured to ssDNA at T d , which subsequently bound to primers at T r and amplied to produce a large amount of products. 15,17 Thus, late cycles were initiated by melting and annealing of the amplicons and intrusion of primers into denaturing bubbles. Based on the primer design strategy of SEA, we designed a pair of ASFV-specic primers targeting the B646L gene with a product length of 42 bp (Table 1).…”

“…The ASEA method has the advantages of sensitivity, rapidity and low cost, and is suitable for large-scale screening of pathogenic microorganisms. 16,17 However, it is worth noting that when it is difficult to design the appropriate primers based on the genome sequence of a specic pathogen, potential by-products such as primer dimers and hairpin structures may lead to the appearance of uorescence curves similar to those of the target, which will interfere with the judgment of results.…”

Real-time monitoring of isothermal nucleic acid amplification on a smartphone by using a portable electrochemical device for home-testing of SARS-CoV-2

“…Although the test strips based on antigen tests have been widely used for at-home SARS-CoV-2 detection at present, negative results of strips need to be interpreted with caution due to the number of false negative tests, with a broad variation in virus concentration [ 3 ]. Compared with immunoassays like antigen tests, nucleic acid amplification tests (NAATs) are considered more powerful tools for pathogen detection [ 4 ], among which real-time fluorescence polymerase chain reaction (PCR) has been employed as the gold standard for SARS-CoV-2 detection due to its excellent sensitivity and specificity [ 5 , 6 ]. However, conventional PCR normally requires bulky and costly thermal cycle and sophisticated fluorescence optical components, leading to the application of this approach only centralized in medical laboratories [ 7 ], while being less suitable for home-testing or in primary medical units lacking funding, which would not benefit to prevent COVID-19 transmission timely or control this acute infectious disease in situ [ 8 ].…”

Nucleic acid extraction without electrical equipment via magnetic nanoparticles in Pasteur pipettes for pathogen detection

“…The MNPs of a commercial kit that adopts traditional MNPs based nucleic acid extraction method were employed to extract nucleic acid of pathogens from same samples via both Pasteur pipette system and this commercial kit. The extracted nucleic acid was directly used as template for accelerated denaturation bubbles mediated strand exchange amplification (ASEA), a rapid and convenient amplification method established by us previously [ 23 , 24 ]. Extraction efficiency of our approach was evaluated by comparing the detection time and sensitivity of ASEA to the target nucleic acid extracted by these two methods.…”