Development and application of the MIRA and MIRA-LFD detection methods of Spiroplasma eriocheiris

Guo, Ying; Zhang, Leiting; Yang, Yue; Li, Jiaying; Luan, Xiaoqi; Gong, Sinan; Ma, Yubo; Gu, Wei; Du, Jie; Meng, Qingguo

doi:10.1016/j.jip.2023.108017

Cited by 2 publications

(4 citation statements)

References 29 publications

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“…Additionally, the PCR and qPCR assays exhibited concordant results for the three genes when compared with MIRA-LFD. Taken together with previous studies on MIRA-LFD, the current study indicates that MIRA-LFD is highly specific for target genes without cross-reactivity with other closely related bacteria [31,33,34].…”

Section: Evaluation Of the Sensitivity And Specificity Of The Tlh Trh...supporting

confidence: 85%

“…and 20 min for V. vulnificus [32,35]. However, recent studies using the MIRA assay have reported that the positive test line could be observed after 5 min for S. eriocheiris, A. baumannii, and Streptococcus agalactiae [33,34,36]. Since the MIRA assay applies a different source of recombinase (Streptomyces azure recA, SC-recA) compared to the RPA assay that uses the RecA/Rad 51 ortholog of bacteriophage T4, T4 UvsX, this may result in different incubation times for amplification [31].…”

Section: Optimization Of Mira-lfd To Amplify Tlh Trh and Tdh Genesmentioning

confidence: 99%

“…Previous studies on the detection of V. parahaemolyticus using RPA assays have reported detection limits ranging from 76 to 2 CFU of bacterial culture and 10 pg of genomic DNA [27][28][29][30]. More recently, MIRA assays for various pathogens have shown detection limits ranging from 760 to 6 CFU of bacterial culture and ranging from 97 pg to 64 fg of genomic DNA [31,33,34,[36][37][38].…”

Section: Evaluation Of the Sensitivity And Specificity Of The Tlh Trh...mentioning

confidence: 99%

“…Previous studies on the detection of V. parahaemolyticus using RPA assays hav ported detection limits ranging from 76 to 2 CFU of bacterial culture and 10 pg of geno DNA [27][28][29][30]. More recently, MIRA assays for various pathogens have shown detec limits ranging from 760 to 6 CFU of bacterial culture and ranging from 97 pg to 64 f genomic DNA [31,33,34,[36][37][38]. Similar detection limit results were obtained with the qPCR assay, which could detect as little as 10 fg of gDNA for the tdh and trh genes and 1 fg of the tlh gene (Figure 6A).…”

Section: Evaluation Of the Sensitivity And Specificity Of The Tlh Trh...mentioning

confidence: 99%

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Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus

Park,

Zhang

2024

Pathogens

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Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.

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Section: Evaluation Of the Sensitivity And Specificity Of The Tlh Trh...supporting

confidence: 85%

Section: Optimization Of Mira-lfd To Amplify Tlh Trh and Tdh Genesmentioning

confidence: 99%

Section: Evaluation Of the Sensitivity And Specificity Of The Tlh Trh...mentioning

confidence: 99%

Section: Evaluation Of the Sensitivity And Specificity Of The Tlh Trh...mentioning

confidence: 99%

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Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus

Park,

Zhang

2024

Pathogens

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Development and evaluation of a real-time multienzyme isothermal rapid amplification assay for rapid detection of Streptococcus pneumoniae

Xing,

Duan,

Jiang

et al. 2024

Sci Rep

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Streptococcus pneumoniae is a significant pathogen causing infectious diseases, including pneumonia, otitis media, septicemia, and meningitis. The introduction of multivalent vaccines has coincided with a remarkable decrease in the number of pneumococcal-related deaths. Despite this, pneumococcal infection remains a significant cause of death among children under 5 years old and adults aged 65 or older at a global level. Therefore, early detection of S. pneumoniae infection is crucial for prognosis of pneumococcal infection patients. In this study, we evaluated the utility of a real-time multienzyme isothermal rapid amplification (MIRA) assay for detecting S. pneumoniae and other non- S. pneumoniae bacterial species. A primer–probe set targeting the S. pneumoniae lytA gene was designed, followed by optimization of parameters for the MIRA assay. At the same time, we validated the real-time MIRA assay for detecting S. pneumoniae using 79 clinical isolates identified by VITEK MS. The results showed a detection sensitivity and specificity of 100%. These results demonstrate that the designed real-time MIRA assay is a promising, rapid, simple, and reliable method for detecting S. pneumoniae infection in resource-limited areas. It has great potential for application in detecting not only S. pneumoniae but also other non- S. pneumoniae bacterial species.

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Development and application of the MIRA and MIRA-LFD detection methods of Spiroplasma eriocheiris

Cited by 2 publications

References 29 publications

Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus

Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus

Development and evaluation of a real-time multienzyme isothermal rapid amplification assay for rapid detection of Streptococcus pneumoniae

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