Establishment of Recombinase Polymerase Amplification assay for rapid and sensitive detection of Orientia tsutsugamushi in Southeast Asia

Nguyen, Chinh Trong; Nguyen, Ung Dinh; Le, Thuy T.; Bui, Hoai Thi; Nguyễn, Anh Ngọc; Trieu, Nguyet Thi; Trieu, Long Phi; Bui, Sy Tien; Nguyen, Chuyen Van; Hoang, Luong Van; Ho, Son Anh; Nguyen, Ba Van; Stenman, Jakob; Ho, Tho Huu

doi:10.1016/j.actatropica.2020.105541

Cited by 4 publications

(3 citation statements)

References 30 publications

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“…Being the common cause of fever in AES and AFI patients, delayed diagnosis of ST may contribute to multiple organ dysfunction and up to a 25% increased mortality rate, specifically in the pediatric population [32]. Hence, a rapid diagnostic test with at least 70% sensitivity is required [33]. The sensitivity of molecular tests, including PCR and real-time PCR, over conventional immunoassay has been well established; nevertheless, they are only available in centralized laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…Based on isothermal detection, i.e., RPA [33,37,38] or LAMP [39][40][41] integrated with either a visual, fluorescent, or lateral flow test of ST, were developed earlier (Table 2). The sensitivity and specificity of these diagnostic approaches made using isothermal amplification alone, either with LAMP or RPA, demonstrated sensitivity ranging from 20 to 96.1% and a specificity ranging from 99.16 to 100% [39][40][41].…”

Section: Discussionmentioning

confidence: 99%

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CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus

Bhardwaj,

Nanaware,

Behera

et al. 2023

Biosensors

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Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus

Bhardwaj,

Nanaware,

Behera

et al. 2023

Biosensors

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show abstract

“…Additionally, 5-end guanines should be avoided while 3-end cytidines and guanines improve amplification performance, while GC content should be between 30% and 70%. Although RPA can work at a range of temperatures from 22-45 • C, optimal temperatures tend to be in the range of 37-42 • C [152,153], and multiple equipment can be employed to regulate the temperature such as incubators, heating blocks, or even body heat, facilitating its use as a point-of-care test in the field [154]. RPA can successfully amplify dsDNA, ssDNA, cDNA, and methylated DNA from a large variety of organisms such as viruses, bacteria, fungi, animals, and plants.…”

Section: Recombinase Polymerase Amplification (Rpa)mentioning

confidence: 99%

Tracking SARS-CoV-2: Novel Trends and Diagnostic Strategies

et al. 2021

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The COVID-19 pandemic has had an enormous impact on economies and health systems globally, therefore a top priority is the development of increasingly better diagnostic and surveillance alternatives to slow down the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In order to establish massive testing and contact tracing policies, it is crucial to have a clear view of the diagnostic options available and their principal advantages and drawbacks. Although classical molecular methods such as RT-qPCR are broadly used, diagnostic alternatives based on technologies such as LAMP, antigen, serological testing, or the application of novel technologies such as CRISPR-Cas for diagnostics, are also discussed. The present review also discusses the most important automation strategies employed to increase testing capability. Several serological-based diagnostic kits are presented, as well as novel nanotechnology-based diagnostic methods. In summary, this review provides a clear diagnostic landscape of the most relevant tools to track COVID-19.

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Rickettsia and Orientia

Blanton,

Walker,

Bouyer

2023

ClinMicroNow

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The family Rickettsiaceae comprises two genera of small, obligately intracellular bacteria that reside free within the host cell's cytosol, namely, Rickettsia and Orientia . Pathogenic Rickettsia spp. reside in an arthropod host for at least a part of their life cycle, during which they are maintained by transovarian transmission and/or cycles involving horizontal transmission to vertebrate hosts. Nucleic acid amplification testing has been applied to the amplification of the DNA of spotted fever group and typhus group rickettsiae, usually from peripheral blood, buffy coat, or plasma and occasionally from fresh, frozen, or paraffin‐embedded tissue and from swabs of the ulcerated bases of eschars. The limited quantities of circulating rickettsial DNA during infection greatly hinder the clinical sensitivity of molecular detection from peripheral blood specimens. Species identification of rickettsial isolates was performed by microimmunofluorescence serotyping. Enzyme immunoassay has become an increasingly used method to detect antibodies reactive against Orientia tsutsugamushi .

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Establishment of Recombinase Polymerase Amplification assay for rapid and sensitive detection of Orientia tsutsugamushi in Southeast Asia

Cited by 4 publications

References 30 publications

CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus

CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus

Tracking SARS-CoV-2: Novel Trends and Diagnostic Strategies

Rickettsia and Orientia

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