A quantitative loop-mediated isothermal amplification assay for detecting a novel goose astrovirus

He, Dalin; Yang, Jing; Jiang, Xiaoning; Lin, Yun; Chen, Hao; Tang, Yi; Diao, Youxiang

doi:10.1016/j.psj.2020.09.077

Cited by 24 publications

(24 citation statements)

References 31 publications

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“…The use of fluorescent labels (e.g. [38]) is also widely used and will be reported later with this Si/BST system, but as highlighted by others, these labels can also cause some inhibition of the enzyme. Thus, in order to compare the activity of the silicaimmobilised BST LF without additives, a two-step analysis was performed using a modified LAMP reaction targeting P. knowlesi 18S rRNA with only the F3/B3 primers from the P.KNO-LAU primer set (Figure S3).…”

Section: Activity Of the Fusion Proteinsmentioning

confidence: 93%

Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

et al. 2022

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Bacillus stearothermophilus large fragment (BSTLF) DNA polymerase is reported, isolated on silica via a fused R5 silica-affinity peptide and used in nucleic acid diagnostics. mCherry (mCh), included in the fusion construct, was shown as an efficient fluorescent label to follow the workflow from gene to diagnostic. The R5 immobilisation on silica from cell lysate was consistent with cooperative R5-specific binding of R52-mCh-FL-BSTLF or R52-mCh-H10-BSTLF fusion proteins followed by non-specific protein binding (including E. coli native proteins). Higher R5-binding could be achieved in the presence of phosphate, but phosphate residue reduced loop-mediated isothermal amplification (LAMP) performance, possibly blocking sites on the BSTLF for binding of β- and γ-phosphates of the dNTPs. Quantitative assessment showed that cations (Mg2+ and Mn2+) that complex the PPi product optimised enzyme activity. In malaria testing, the limit of detection depended on Plasmodium species and primer set. For example, 1000 copies of P. knowlesi 18S rRNA could be detected with the P.KNO-LAU primer set with Si-R52-mCh-FL-BSTLF , but 10 copies of P. ovale 18S rRNA could be detected with the P.OVA-HAN primer set using the same enzyme. The Si-immobilised BSTLF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R52-mCh-FL-BSTLF production was transferred from Cambridge to Accra and set up de novo for a trial with clinical samples. Different detection limits were found, targeting the mitochondrial DNA or the 18S rRNA gene for P. falciparum. The results are discussed in comparison with qPCR and sampling protocol and show that the Si-BSTLF polymerase can be optimised to meet the WHO recommended guidelines. Graphical abstract

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Section: Activity Of the Fusion Proteinsmentioning

confidence: 93%

Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

et al. 2022

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“…The rapid detection relies on specific primers of GAstV ORF2 gene in the reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay [ 38 ], TaqMan-based one-step real-time RT-PCR assay [ 39 ], and SYBR Green I real-time PCR assay [ 40 ]. There are also virus detection methods based on GAstV ORF1a [ 41 ] and ORF1b [ 42 ]. Advantages of all these molecular detection methods include simplicity, rapid performance, sensitivity, and high specificity, which makes them valuable in epidemiological research.…”

Section: Separation and Detection Of Gastvmentioning

confidence: 99%

A Review of Emerging Goose Astrovirus Causing Gout

Liu

Sun

Liao

2022

BioMed Research International

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In recent years, an infection in geese caused by goose astrovirus (GAstV) has repeatedly occurred in coastal areas of China and rapidly spread to inland provinces. The infection is characterized by joint and visceral gout and is fatal. The disease has caused huge economic losses to China’s goose industry. GAstV is a nonenveloped, single-stranded, positive-sense RNA virus. As it is a novel virus, there is no specific classification. Here, we review the current understanding of GAstV. The virus structure, isolation, diagnosis and detection, innate immune regulation, and transmission route are discussed. In addition, since GAstV can cause gout in goslings, the possible role of GAstV in gout formation and uric acid metabolism is discussed. We hope that this review will inform researchers to rapidly develop effective methods to prevent and treat this disease.

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“…10 For example, the LAMP assay has reported quantifiable DNA in the range of 10−10 7 copies/μL. 11 However, the current INAAT approaches rely on the time to the onset of amplification for quantification, thus requiring real-time monitoring. Further, rapid amplification requires tight temporal control in such assays, and rapid generation of spurious nonspecific products mandates the use of specific probes.…”

Section: ■ Introductionmentioning

confidence: 99%

Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Valloly

Roy

2022

Anal. Chem.

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Amplification-based quantitative polymerase chain reaction (qPCR) provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to many settings. Quantitative isothermal amplification methods alleviate the need for thermal cyclers; however, they still require continuous monitoring of the nucleic acid amplification on sophisticated readers. Here, we adapted an isothermal recombinase polymerase amplification (RPA) reaction to develop a semiquantitative method that relies on the final amplicon yield to estimate the initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and showed that the amplicon yields at the end of the RPA reaction correlated well with the starting DNA concentration while reducing nonspecific amplification robustly. We demonstrate this approach, termed quantitative endpoint RPA (qeRPA), to detect DNA over five log orders with a detection limit of 100 molecules. Using a linear regression model of the normalized endpoint intensity (NEI) standard curve, we estimate the viral load from the serum of dengue virus-infected patients with comparable performance to qPCR. Unlike the conventional isothermal quantitative methods, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field deployment in resource-limited settings.

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A quantitative loop-mediated isothermal amplification assay for detecting a novel goose astrovirus

Cited by 24 publications

References 31 publications

Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis

A Review of Emerging Goose Astrovirus Causing Gout

Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

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