New approach for the identification of potentially toxigenic Corynebacterium sp. using a multiplex PCR assay

Sunarno, Sunarno; Khariri,; Muna, Fauzul; Sariadji, Kambang; Rukminiati, Yuni; Febriyana, Dwi; Febrianti, Tati; Saraswati, Ratih Dian; Susanti, Ida; Puspandari, Nelly; Karuniawati, Anis; Malik, Amarila; Soebandrio, Amin

doi:10.1016/j.mimet.2021.106198

Cited by 5 publications

(11 citation statements)

References 28 publications

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“…We used the tox gene as a target to identify toxigenicity, similar to their study. Unlike our previous study (13), we did not identify the NTTB strains to reduce the cost in this study because of the high cost of the PCR probe. Furthermore, previous studies documented that NTTB strains were never observed in diphtheria samples in Indonesia (4,10,13).…”

Section: Discussionmentioning

confidence: 96%

“…The established conventional PCR assay was carried out to ensure the accuracy of the multiplex real-time PCR assay. The procedure of the conventional PCR assay was according to the protocol described in the previous study (13). The multiplex real-time PCR results were considered accurate when the results were similar to the established conventional PCR results, even though the conventional methods showed different results.…”

Section: Established Conventional Pcr and Data Analysismentioning

confidence: 99%

“…However, PCR usually cannot be used to distinguish between toxigenic and non-toxigenic tox gene bearing (NTTB) strains. We previously succeeded in developing PCR to predict the NTTB strains; however, it is still limited to two types, namely base-25 deletion or base-55 deletion NTTB (13).…”

Section: Introductionmentioning

confidence: 99%

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Development and Application of Dtxr and Tox Genes Targeting Real-time PCR to Identify Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis Simultaneously

Sunarno¹,

Hartoyo²,

Amalia³

et al. 2022

Jundishapur J Microbiol

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Background: Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis are known as diphtheria-causing bacteria. Although diphtheria therapy is administered based on the clinical manifestations, some cases are mild and atypical. The immediate and accurate identification of diphtheria-causing bacteria is of paramount importance to prevent the spread of the disease and provide case management as early as possible. Unfortunately, conventional methods as the gold standard are time-consuming. Objectives: This study aimed to develop and implement a multiplex real-time PCR with the dtxR and tox genes as the target to identify three species of diphtheria-causing bacteria and screen their toxigenicity quickly and accurately. Methods: The research sample encompassed seven reference strains, one synthetic DNA, 30 archived isolates, and 924 clinical specimens isolated from 311 diphtheria cases and 613 close contacts. The conventional methods as the gold standard and the established PCR assay were used to verify the results of multiplex real-time PCR developed in this study. Results: The multiplex real-time PCR could identify seven reference strains, one synthetic DNA, and 30 archived isolates as accurately as the conventional methods and the established PCR. Similar to established PCR, the multiplex real-time PCR identified diphtheria-causing bacteria in 120 (38.6%) out of 311 and 12 (2%) out of 613 clinical specimens from diphtheria cases and close contacts, respectively. Meanwhile, the conventional methods identified diphtheria-causing bacteria in 79 (25.4%) out of 311 and three (0.5%) out of 613 clinical specimens. Conclusions: The multiplex real-time PCR developed in this study can be used to identify three species of diphtheria-causing bacteria and screen their toxigenicity quickly and accurately. However, in this study, nodiphtheria-causing bacteria other than C. diphtheriae was found in the clinical samples using the PCR or conventional methods. PCR is more sensitive than the conventional methods and can be used as an additional test in diphtheria laboratories.

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Section: Discussionmentioning

confidence: 96%

Section: Established Conventional Pcr and Data Analysismentioning

confidence: 99%

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Development and Application of Dtxr and Tox Genes Targeting Real-time PCR to Identify Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis Simultaneously

Sunarno¹,

Hartoyo²,

Amalia³

et al. 2022

Jundishapur J Microbiol

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“…The tox and dtxR genes are commonly used in laboratory tests for diphtheria using PCR assays. We sought to develop PCR assays with the tox and dtxR genes as targets for species identification and toxigenicity, including predicting 2 types of NTTB, resulting in an improved method [4] . Here, we present the whole-genome sequencing (WGS) data of 18 C. diphtheriae isolates from Indonesia ( Table 1 ).…”

Section: Data Descriptionmentioning

confidence: 99%

“…The isolates were collected since 2012 until 2015, mostly have mitis subtype (61%). We used these data to identify SNPs associated with the tox and dtxR genes to verify the accuracy of the PCR assay [4] . We also used these data for molecular typing using the MLST approach [5] .…”

Section: Data Descriptionmentioning

confidence: 99%

Whole-genome sequencing data of Corynebacterium diphtheriae isolated from diphtheria outbreaks in Indonesia

et al. 2022

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Challenges of Diphtheria Toxin Detection

Prygiel,

Mosiej,

Polak

et al. 2024

Toxins

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Diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Moreover, new Corynebacterium species with the potential to produce diphtheria toxin have also been described. Therefore, the detection of the toxin is the most important test in the microbiological diagnosis of diphtheria and other corynebacteria infections. Since the first demonstration in 1888 that DT is a major virulence factor of C. diphtheriae, responsible for the systemic manifestation of the disease, various methods for DT detection have been developed, but the diagnostic usefulness of most of them has not been confirmed on a sufficiently large group of samples. Despite substantial progress in the science and diagnostics of infectious diseases, the Elek test is still the basic recommended diagnostic test for DT detection. The challenge here is the poor availability of an antitoxin and declining experience even in reference laboratories due to the low prevalence of diphtheria in developed countries. However, recent and very promising assays have been developed with the potential for use as rapid point-of-care testing (POCT), such as ICS and LFIA for toxin detection, LAMP for tox gene detection, and biosensors for both.

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New approach for the identification of potentially toxigenic Corynebacterium sp. using a multiplex PCR assay

Cited by 5 publications

References 28 publications

Development and Application of Dtxr and Tox Genes Targeting Real-time PCR to Identify Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis Simultaneously

Development and Application of Dtxr and Tox Genes Targeting Real-time PCR to Identify Corynebacterium diphtheriae, C. ulcerans, and C. pseudotuberculosis Simultaneously

Whole-genome sequencing data of Corynebacterium diphtheriae isolated from diphtheria outbreaks in Indonesia

Challenges of Diphtheria Toxin Detection

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