Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis

Nguyen, Anh Thi Ngoc; Bui, Hoai Thi; Pham, Quynh Thi-Huong; Hoang, Ly Thi Thao; Ta, Hung Xuan; Heikkinen, Timo; Le, Duyet Van; Van, Trang Dinh; Ngo, Nam Quoc; Huynh, Phuong Thi Hong; Tran, Trang Thi Huyen; Phan, Hoan Quoc; Hoang, Luong Van; Doorn, H. Rogier van; Nguyen, Diep Thi Ngoc; Nguyên, Tam; Vo, Nam S; Vo, Cuong Viet; Trinh, Sau Khac; Pham-The, Tai; Le, Quang Duc; Le, Phan Van; Nguyen, Son Thai; Tran, Loan Thi; Vu, Toan Dinh; Vu, Quynh Anh Nguyen; Trieu, Nguyet Thi; Le, Thuy T.; Nguyen, Ung Dinh; Steman, Jakob; Ho, Tho Huu

doi:10.1016/j.cca.2022.04.997

Cited by 4 publications

(2 citation statements)

References 29 publications

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“…The positive samples of C. brachyotis were detected in guano samples and rectal swabs collected from Singapore Zoological Garden and Sungei Buloh Wetland Reserve. Although they used a nested PCR protocol (Drosten et al., 2003), their positivity rate might be low due to their primers sensitivity, unlike our study used the degenerate primers and semi‐nested PCR protocol and it is known that this protocol has a higher sensitivity rate and specificity in individual testing (Dong et al., 2022; Thi Nguyen et al., 2022).…”

Section: Discussionmentioning

confidence: 87%

Detection of novel coronaviruses from dusky fruit bat (Penthetor lucasi) in Sarawak, Malaysian Borneo

Habeebur‐Rahman,

Noni,

Khan

et al. 2023

Veterinary Medicine & Sci

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BackgroundSarawak has one of the highest diversity of fruit bats species (family Pteropodidae) in Malaysia, with 19 species described. Most coronavirus (CoV) studies have mainly focused on insectivorous bats, resulting in a lack of information on CoVs present in frugivorous bats. In addition, bat CoV surveillance activities are lacking in Malaysia.ObjectivesOur study focuses on determining the presence of bat CoVs in dusky fruit bat (Penthetor lucasi).MethodsGuano samples belonging to P. lucasi were collected from Wind Cave Nature Reserve. The samples were screened for the presence of CoVs using validated hemi‐nested consensus RNA‐dependent RNA polymerase consensus primers.ResultsThe bat CoV positivity rate was 38.5% (n = 15/39), with the viruses belonging to two subgenera: Alphacoronavirus (α‐CoV) and Betacoronavirus (β‐CoV). Phylogenetic analysis revealed that CoVs from 14 samples of P. lucasi belong to the genus α‐CoV and may represent previously described genetic lineages in insectivorous bats in Wind Cave. However, only one sample of P. lucasi was detected with β‐CoV which is closely related to subgenus Nobecovirus, which is commonly seen in frugivorous bats.ConclusionsThis study provides the first available data on CoVs circulating in P. lucasi.

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Section: Discussionmentioning

confidence: 87%

Detection of novel coronaviruses from dusky fruit bat (Penthetor lucasi) in Sarawak, Malaysian Borneo

Habeebur‐Rahman,

Noni,

Khan

et al. 2023

Veterinary Medicine & Sci

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“…When used with Semi-nested RT-PCR and the VP1 gene, Semi-nested PCR is hitting the target specifically and quickly. Researchers can depend on it to identify the incriminated strain, especially with viruses such as RNA Viruses, which contains many different strains [28]. This is the first reported use of Semi-nested RT-PCR for identifying the SAT2 serotype in buffaloes in Iraq, further highlighting its potential as a valuable diagnostic tool in FMD surveillance and control programs.…”

Section: Discussionmentioning

confidence: 95%

Clinical and Molecular Detection of Foot and Mouth Disease in Buffalo, Iraq

Enad,

Mansour

2025

Egyptian Journal of Veterinary Sciences

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OOT-AND-MOUTH disease is a transboundary, highly contagious viral disease. The A, Asia 1, and O are the main serotypes reported in Iraq over the last 60 years, however, a recent outbreak revealed a different phenomenon. So, this study aims to detect the serotype(s) of FMDV that caused the most recent disease outbreak in buffaloes in Wassit and Dhi-Qar, Iraq. This will be done by using both clinical and molecular methods, and the confirmed serotype will come from phylogenetic analysis. In total, 70 buffaloes of different genders and ages were selected for the current study, performed during August and September 2023. All animals in this study were examined clinically to determine the suspected infected animals and then subjected to the collection of epithelial tissue samples. Clinically, the infected buffaloes showed different signs, such as fever, reduced appetite, ulcers in the mouth and foot, and impaired mobility. This study used (Semi-Nested RT-PCR) to detect the SAT2 serotype in buffaloes. The findings indicated that 61 out of 70 local isolates (87.14 percent) tested positive for SAT2. These isolates were subsequently submitted to GenBank and assigned accession numbers ranging from (OR576814.1 -OR576820.1). As the first recorded Iraqi SAT2 isolate. The Iraqi isolates were clustered in the same branch as the Egyptian isolate (MZ097480.1). This study is the first to implement the Semi-nested RT-PCR technique for identifying the SAT2 serotype in buffaloes in Iraq. The findings of this study have the potential to provide valuable insights into the formulation and implementation of FMD protection programmers in Iraq.

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