Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae

Abstract

Haemophilus influenzae is an opportunistic pathogen usually causing bacteremia, meningitis, and pneumonia in children. Here, we developed a method based on multiple cross displacement amplification (MCDA) method and real-tme fluorescence technique for rapid detection of H. influenzae. A set of 10 primers was designed for the H. influenzae real-time MCDA reaction, and a core primer was modified with a restriction endonuclease recognition sequence, a fluorescent, and a quencher according to the principle of the real-time MCDA assay. The H. influenzae real-time MCDA reactions were performed using a fluorescence instrument at 63°C for 40 min. The H. influenzae real-time MCDA assay can specifically detect H. influenzae without any cross-reaction with other bacteria as our results confirmed. The sensitivity of our assay is as low as 10 CFU per reaction. To validate its feasibility, our assay was applied to 40 DNA extracted from sputum samples. The results obtained from H. influenzae real-time MCDA were compared with that of H. influenzae–loop-mediated isothermal amplification (H. influenzae-LAMP) and MCDA-based lateral flow biosensor (MCDA-LFB). The positive rate of the real-time MCDA assay was 62.5%, which was consistent with the H. influenzae-MCDA-LFB assay, but was more sensitive than H. influenzae-LAMP (57.5%). Furthermore, the H. influenzae real-time MCDA assay takes only 40 min, which was less than that of a traditional PCR test. Taken together, the H. influenzae real-time MCDA assay reported here offers a new and valuable diagnostic tool for the reliable and rapid detection of H. influenzae.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing

“…When analyzing with a pseudotyped virus, the LoD level reached down to 12.5 copies per microliter. Although the sensitivity of multiplex ET-PCR assay was marginally lower than that of previous isothermal amplication-based studies, such as MCDA, 26 LAMP, 24 and RPA 36 techniques, as well as the established real-time PCR methods, 19,20 the newly developed method was able to identify generic, clade I and clade II MPXV strains in a single reaction rather than one or two of them. In addition, the combination of generic and clade-specic testing improves detection accuracy and aids in new clade identication.…”

“…9 Thus, NAATs are preferred laboratory tests to identify and characterize MPXV with rapidness, high sensitivity and specicity. 16 Recently, many NAATs have been developed to conrm MPXV infection, including conventional PCR, 17,18 realtime PCR, [19][20][21] recombinase polymerase amplication (RPA), 22,23 loop-mediated isothermal amplication (LAMP), 24,25 multiple cross displacement amplication (MCDA), 26 and restriction length fragment polymorphism (RFLP), 10,27 all of which commonly target one specic region of the monkeypox virus genome and possess varying sensitivities and limits of detection (LoD) for MPXV diagnosis. 28 Although the whole genome sequencing technique has also been reported to be deployed for MPXV strain identication, it mainly occurred in centralized and specialized laboratories, 29,30 which were usually not available for large-scale and timely testing.…”

Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

“…The MTB-rt-MCDA assay developed here was a MCDA-based detection platform using MCDA technique for target amplification and real-time fluorescence detector for result reporting. As a rapid, simple and economical amplification method, MCDA technique has more and more applications to diagnose bacteria, viruses and fungi infections [ [18] , [19] , [20] , [21] ]. Due to its simplicity, convenience and low cost, MCDA reactions could be easily conducted under an isothermal condition provided by a thermostatic heater or a water bath.…”

Rapid detection of human cytomegalovirus by multienzyme isothermal rapid amplification and lateral flow dipsticks

“…These include multiple cross displacement amplification (MCDA), loop mediated isothermal amplification (LAMP), cross primer amplification (CPA), strand displacement amplification (SDA), helicase dependent amplification (HDA), recombinant enzyme polymerase amplification (RPA), polymerase spiral response (PSR), and others. For MCDA, LAMP, and CPA ( Fang et al., 2009 ; Li et al., 2019 ; Sun et al., 2022 ), the primer design procedure is extremely complicated, and the amplicon validation stage is also challenging. SDA has a low amplification efficiency for long targets and requires sample preparation ( Yan et al., 2014 ; Wang et al., 2022 ).…”