Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B

Cao, Cuiming; Zhang, Fang; Ji, Mingyu; Pei, Fengyan; Fan, Xiaojun; Shen, Hong; Wang, Qingxi; Yang, Weihua; Wang, Yunshan

doi:10.1016/j.toxicon.2016.03.019

Cited by 8 publications

(17 citation statements)

References 42 publications

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“…ORF1ab gene, E gene and N gene were selected and primers for them were designed. We have used the RT-LAMP method to detect pathogenic microorganisms and achieved good results(9, 10). In 2003, RT-LAMP had been reported as a quick and easy diagnostic method for SARS coronavirus(11, 12).…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

Yang

Dang

Wang

et al. 2020

Preprint

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Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real- time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

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Section: Introductionmentioning

confidence: 99%

Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

Yang

Dang

Wang

et al. 2020

Preprint

Self Cite

128

125

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“…The graph was generated as early as 15 min in the present study, which was also supported by the findings observed in other methods as shown in Figure 2. The time for speB gene detection in the present study was shorter than that of the colorimetry method used by Cao et al, which was 35 min (16). In their study, the LAMP method was applied, but the endpoint detection for the LAMP reaction was only based on the color change by naked eye observation (16).…”

Section: Discussionmentioning

confidence: 64%

“…Primers established by Cao et al were used for the preliminary screening of speB gene detection (16). The PCR assays of 43 S. pyogenes clinical isolates were performed by using DNA Mini Kit (QIAGEN Company, Germany).…”

Section: Pcr Assay For Preliminary Detection Of Streptococcal Pyrogenic Exotoxin B (Speb) Genementioning

confidence: 99%

“…The genomic DNA concentration was measured using a Micro-Spectrophotometer (Nanodrop ND-1000, Bio-Rad, USA) and was adjusted to 100 ng/µL with sterile distilled water. This was followed by serial dilution from 102 ng/µL to 10-6 ng/ µL based on the established protocols (16). In addition, similar steps were performed for the PCR assay.…”

Section: Specificity and Sensitivity Of Speb Detection By The Rt-lamp Assaymentioning

confidence: 99%

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Improved Visual Detection of speB Gene in Streptococcus pyogenes Isolates by Real-time Loop-Mediated Isothermal Amplification Turbidimetry Method

Simon

Subbiah

Than

et al. 2021

Jundishapur J Microbiol

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Background: Group A Streptococcus (GAS) causes a wide array of clinical manifestations ranging from mild pharyngitis to suppurative and non-suppurative severe debilitating diseases. Hence, a simple, rapid detection method with high sensitivity and specificity is needed. Objectives: This study embarked on the visual detection of the streptococcal pyrogenic exotoxin B (speB) gene by Real-time Turbidimetry and Loop-mediated Isothermal Amplification (RT-LAMP) methods. The real-time monitoring of the sigmoidal graph generated from a turbidometry method was incorporated in the assay. Methods: The amplification of the speB gene was virtually observed in real-time monitoring of the graph (sigmoidal curve) generated via a turbidimeter, thus providing a “guide” to accurately estimate the time to positivity for the gene detection. Results: The targeted gene was detected at 15 min but was optimally amplified within 45 min at an isothermal temperature of 63°C with 100% specificity using an established set of primers. The formation of sigmoidal curves was correlated with other visual observations by the naked eye (from orange to green), ultra-violet light (green fluorescence), and agarose gel electrophoresis. The improved detection limit of the real-time RT-LAMP assay was also observed compared to conventional PCR assay (0.001 pg/µL versus 1 ng/µL). Conclusions: In conclusion, this improved visual detection of RT-LAMP assay could provide additional insight for rapid, cost-effective, and reliable identification of GAS via speB gene detection in low or middle-income countries. It could also be a very important tool to improve the healthcare management of patients infected with GAS in the future.

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“…ORF1ab gene, E gene and N gene were selected and primers for them were designed. We have used the RT-LAMP method to detect pathogenic microorganisms and achieved good results (9,10). In 2003, RT-LAMP had been reported as a quick and easy diagnostic method for SARS coronavirus (11,12).…”

mentioning

confidence: 99%

Rapid and accurate detection of three genes in SARS-CoV-2 using RT-LAMP method

Yang

Dang

Wang

et al. 2020

Preprint

Self Cite

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Background Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. It has the characteristics of rapid transmission, difficult treatment, rapid deterioration, and high mortality rate. Reliable and rapid diagnosis of SARS-CoV-2 infection is critical to treat patients and control transmission in time. Methods To detect SARS-CoV-2 nucleic acid faster and more convenient, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. ORF1ab gene, E gene and N gene were selected as the target genes, because ORF1ab gene is the most specific and N gene is the most sensitive, while E gene has both sensitivity and specificity. Over thirty primer sets for them were designed, then the primer sets with rapid response and high specificity were screened out and their loop primers were designed and selected in order to improve the reaction speed further. Using these primers, RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be judged by naked eyes through color change within 30 minutes. Finally, the optimal primer set for each gene was verified with over 208 clinical samples. Results ORF1ab gene, E gene and N gene were detected simultaneously by this method. ORF1ab gene has high specificity and the same sensitivity as RT-PCR currently used in clinic. Although N gene is less specific than ORF1ab, it is 80 times more sensitive than ORF1ab. The sensitivity and specificity of E gene are between them. Simultaneous detection of these three genes can neither cause false positives nor miss samples with low virus concentration, ensuring the sensitivity and specificity of SARS-CoV-2 detection. BLAST comparing results showed the primers for the three genes were highly specific for SARS-CoV-2. And the sequencing results of RT-LAMP products showed that the amplified fragments were unique to SARS-CoV-2. The accuracy of RT-LAMP assay was 99% as detecting 208 clinical specimens. Conclusions The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

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Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B

Cited by 8 publications

References 42 publications

Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

Improved Visual Detection of speB Gene in Streptococcus pyogenes Isolates by Real-time Loop-Mediated Isothermal Amplification Turbidimetry Method

Rapid and accurate detection of three genes in SARS-CoV-2 using RT-LAMP method

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