Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

Kamboj, Aman; Pateriya, Atul Kumar; Mishra, Anamika; Ranaware, P.B.; Kulkarni, D. D.; Raut, Ashwin Ashok

doi:10.1155/2014/496219

Cited by 10 publications

(4 citation statements)

References 10 publications

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“…The complete list of published molecular assays published until December 2018 includes 2 single round PCR [34,35], 6 nested PCR [22,36,37,38,39], 12 real-time PCR [40,41,42,43,44,45,46,47,48,49,50,51], 1 LAMP [52]; and 1 RPA (Recombinase Polymerase Amplification) [53]. All assays are reported in Table 1 and in Supplementary Table S1.…”

Section: Resultsmentioning

confidence: 99%

Geographical Variability Affects CCHFV Detection by RT–PCR: A Tool for In-Silico Evaluation of Molecular Assays

Gruber¹,

Bartolini²,

Castilletti³

et al. 2019

Viruses

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The Crimean–Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient’s travel history and geographic distribution of the different CCHFV strains.

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Section: Resultsmentioning

confidence: 99%

Geographical Variability Affects CCHFV Detection by RT–PCR: A Tool for In-Silico Evaluation of Molecular Assays

Gruber¹,

Bartolini²,

Castilletti³

et al. 2019

Viruses

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“…Zahraei et al also obtained 100% specificity and less than 20 copies/reaction sensitivity using SybrGreen (34). However, in a study by Kamboj et al, although they obtained a LOD of 5 copies/reaction due to 3' end primer mismatches, the real-time RT-PCR could not identify all 18 clinical samples (35). On the other hand, another investiga- In several studies with probes, specificity was 100%, and sensitivity was high (37,38).…”

Section: Discussionmentioning

confidence: 98%

TaqMan Multiplex Real-time PCR Assay for Crimean-Congo Hemorrhagic Fever Virus Diagnosis Using Armored RNA Technology

Jamehdor

Hosseinirouzbahani

Hoseinishokoh

et al. 2023

Jundishapur J Microbiol

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Background: Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly lethal virus that causes hemorrhagic fever in humans and is endemic in many countries, including Iran. Therefore, fast, accurate, and reliable diagnosis is crucial for patient management and outbreak control. Objectives: This study aims to optimize a TaqMan multiplex real-time RT-PCR for the rapid and specific diagnosis of CCHFV. Methods: In this study, the L (NC_005301.3) and S (NC_005302.1) fragments were used as reference sequences for blast analysis. The L and S sequence segments of CCHFV with more than 90% identity from different areas were downloaded from the Genbank database. Primers and probes were designed based on the best-conserved regions of CCHFV L and S sequence segments. To construct the plasmid, a 1751 bp fragment from the MS2 phage that was previously amplified using cloning primers was inserted into the pET-32a plasmid. The S and L segments of the CCHFV, which were 110 bp and 135 bp, respectively, were inserted downstream of the MS2 phage sequence from HindIII to NotI. The Viral-like particles (VLPs) were produced in Escherichia coli, strain BL-21(DE3), in the presence of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The stability of VLP particles was confirmed in the presence of the ribonuclease enzyme. The fabrication of VLPs was approved by transmission electron microscopy (TEM) with negative staining (1% phosphotungstic acid). To validate the specificity of the primers and probes sequences, we compared them to the NCBI database and tested them experimentally using extracted DNA and RNA samples from healthy subjects and an infectious panel. Results: The VLPs showed complete resistance in the presence of the ribonuclease enzyme, and the TEM results confirmed that the VLPs were correctly produced. The TaqMan multiplex real-time RT-PCR confirmed that the primers and probes were designed correctly and were completely specific to the CCHFV. The limit of detection (LOD) of the multiplex assay for the L and S genes was one copy of the VLPs per µL. Conclusions: This TaqMan assay is reliable for amplifying CCHFV due to its design on conserved regions of the CCHFV sequences, which have minimal variability and high specificity.

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“…Nucleic acid amplification tests. Many nucleic acid amplification tests have been published for CCHFV diagnosis (Table 1), although few tests are commercially available (7,9,11,37,(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58). Reverse-transcription PCR (RT-PCR) is commonly used for the diagnosis of CCHVF in the first 10 to 12 days after symptom onset and is generally more widely available than viral culture (34,37).…”

Section: Assays For Viral Detectionmentioning

confidence: 99%

Diagnostic Testing for Crimean-Congo Hemorrhagic Fever

Raabe

2020

J Clin Microbiol

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Crimean-Congo hemorrhagic fever is the most geographically widespread tick-borne virus, with infection resulting in mortality in up to 30% of cases. Clinical diagnosis alone is difficult due to the nonspecific nature of symptoms; therefore, laboratory diagnostics should be utilized for patients with residence in or travel to regions of endemicity in whom the disease is suspected. This minireview provides an overview of laboratory tests available for Crimean-Congo hemorrhagic fever (CCHF) and their utility in diagnosis with a focus on diagnosing CCHF in humans.

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Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

Cited by 10 publications

References 10 publications

Geographical Variability Affects CCHFV Detection by RT–PCR: A Tool for In-Silico Evaluation of Molecular Assays

Geographical Variability Affects CCHFV Detection by RT–PCR: A Tool for In-Silico Evaluation of Molecular Assays

TaqMan Multiplex Real-time PCR Assay for Crimean-Congo Hemorrhagic Fever Virus Diagnosis Using Armored RNA Technology

Diagnostic Testing for Crimean-Congo Hemorrhagic Fever

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