scholarly journals A miniPCR-duplex Lateral Flow Dipstick Platform in Combination with the Microfluidic Device for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

2021 ◽  
Author(s):  
Achinya Phuakrod ◽  
Witsaroot Sripumk ◽  
Wutthinan Jeamsaksiri ◽  
Pattaraluck Pattamang ◽  
Sumart Loymek ◽  
...  
Keyword(s):  
Lymphatic Filariasis ◽  
Species Identification ◽  
Microfluidic Device ◽  
Lateral Flow ◽  
Detection Accuracy ◽  
Diagnostic Assay ◽  
Sequence Alignments ◽  
Blood Samples ◽  
Lateral Flow Dipstick ◽  
Visual Diagnosis

Abstract Background: Lymphatic filariasis is a major neglected tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. Accordingly, access is now needed to a cost-efficient, non-laborious diagnostic assay(s) capable of detecting low levels of microfilariae and without the need for infrastructure and expensive equipment.Methods: We developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD) assay. The design of the PCR primers was guided by sequence alignments of the HhaI and SspI genes of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection, accuracy, and cross-reactivity of the assay were evaluated, after which the assay was deployed to assess infection using blood samples obtained from Myanmar study participants living along the Thailand-Myanmar border, a region in Tak province endemic W. bancrofti. In addition, blood samples were provided by from Thais in Narathiwat province residing in an area endemic for brugian filariasis. We also combined a previously reported semi-automated microfluidic device with the miniPCR-DLFD to facilitate rapid detection and species identification of microfilariae in human blood.Results: The miniPCR-DLFD assay exhibited a detection limit of two microfilariae per mililiter blood sample, and cross-amplification was not observed with from other parasites. For field validation, microfilariae and DNA of W. bancrofti were detected from two (0.6%) and five (1.5%) out of 328 (100%) blood samples, respectively. For the Narathiwat samples, microfilariae and DNA of B. malayi were detected from one (0.46%) and two (0.9%) out of 216 (100%) blood samples, respectively. A rapid and visual of species identification was accurately obtained in all microfilariae entrapped by our previously developed microfluidic device.Conclusion: A miniPCR-DLFD platform alone or coupled with the microfluidic device provided rapid and visual diagnosis of lymphatic filariasis. The microfluidic device, the miniPCR, and the DLFD are all portable. Coupled with a portable microcentrifuge for DNA extraction, this is a promising platform for the diagnosis of lymphatic filariasis in point-of-collection settings with a modest cost (~USD $ 5) per sample.

scholarly journals A miniPCR-Duplex Lateral Flow Dipstick Platform for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

Diagnostics ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1855
Author(s):  
Achinya Phuakrod ◽  
Witsaroot Sripumkhai ◽  
Wutthinan Jeamsaksiri ◽  
Pattaraluck Pattamang ◽  
Sumat Loymek ◽  
...  
Keyword(s):  
Lymphatic Filariasis ◽  
Dna Sequences ◽  
Wuchereria Bancrofti ◽  
Pcr Primers ◽  
Lateral Flow ◽  
Thick Blood Smear ◽  
Noncoding Dna ◽  
Lateral Flow Dipstick ◽  
Visual Diagnosis

Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.

scholarly journals Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Zhuo-ran Li ◽  
Zhen-xing Yang ◽  
Zhan-hong Li ◽  
Xiang Gao ◽  
Zhong-yan Hu ◽  
...  
Keyword(s):  
Disease Virus ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Blood Samples ◽  
Qrt Pcr ◽  
Lateral Flow Dipstick ◽  
Co Detection ◽  
Haemorrhagic Disease ◽  
Analytical Sensitivities

Abstract Background Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. Results In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. Conclusions This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

scholarly journals A Novel Rapid Visual Detection of Toxoplasma Gondii by Combining Recombinase Polymerase Amplification and Lateral Flow Dipstick Coupled With CRISPR-Cas13a Fluorescence Assay

2021 ◽  
Author(s):  
Jinhong Zhao ◽  
Yuanyuan Li ◽  
Qiqi Xue ◽  
Zhiwei Zhu ◽  
Minghui Zou ◽  
...  
Keyword(s):  
Visual Detection ◽  
High Specificity ◽  
Lateral Flow ◽  
Parasitic Disease ◽  
Blood Samples ◽  
Naked Eye ◽  
Detection Assay ◽  
Lateral Flow Dipstick ◽  
Sophisticated Equipment ◽  
Positive Rate

Abstract Background: Toxoplasmosis caused by infecting with Toxplasma gondii is a kind of parasitic disease that prevalent all over the world and does great harm to pregnant women and newborns. Effective, rapid and accurate diagnosis T. gondii is urgently needed to prevent and treatment the toxoplasmosis. The purpose of this study was to develop a rapid visual detection assay using recombinase aided amplification (RAA) and lateral flow dipstick (LFD) coupled with CRISPR-Cas13a fluorescence, henceforth RAA-Cas13a-LFD, for detection of T. gondii.Methods: Targeting 529bp gene of T. gondii, the primers and probes for RAA-Cas13a-LFD assay were designed and screened. The reaction time of RAA-LFD-Cas13a assay was optimized, as well as the sensitivity and specificity was further validated. Finally, the diagnostic performance of T. gondii was evaluated using the RAA-Cas13a-LFD assay for clinical blood samples.Results: The RAA-Cas13a-LFD assay was performed in an incubator block at 37℃ within 2h, and the amplicons were visible through LFD for naked eye visualization. The detection limit of the developed RAA-Cas13a-LFD assay was 1×10-6 ng/μL with high specificity for T. gondii. Compared with qPCR assay, there was a consistent positive rate among the clinical blood samples. Conclusion: In this study, A rapid and visual RAA-Cas13a-LFD assay was developed. It requires no sophisticated equipment and shows promise for on-site surveillance of T. gondii.

scholarly journals Bushmeat Species Identification: Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow (LF) Strip for Identification of Formosan Reeves’ Muntjac (Muntiacus reevesi micrurus)

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 426
Author(s):  
Yun-Hsiu Hsu ◽  
Wei-Cheng Yang ◽  
Kun-Wei Chan
Keyword(s):  
Species Identification ◽  
Operating Time ◽  
Meat Products ◽  
Reaction System ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Food Allergies ◽  
Mitochondrial Cytochrome B ◽  
Meat Species ◽  
A New Technique

The identification of animal species of meat in meat products is of great concern for various reasons, such as public health, religious beliefs, food allergies, legal perspectives, and bushmeat control. In this study, we developed a new technique to identify Formosan Reeves’ muntjac in meat using recombinase polymerase amplification (RPA) in combination with a lateral flow (LF) strip. The DNA extracted from a piece of Formosan Reeves’ muntjac meat was amplified by a pair of specific primers based on its mitochondrial cytochrome b gene for 10 min at a constant temperature ranging from 30 to 45 °C using RPA. Using the specific probe added to the RPA reaction system, the amplified products were visualized on the LF strip within 5 min. The total operating time from quick DNA extraction to visualizing the result was approximately 30 min. The RPA-LF system we designed was efficient when using boiled, pan-fried, roasted, stir-fried, or stewed samples. The advantages of simple operation, speediness, and cost-effectiveness make our RPA-LF method a promising molecular detection tool for meat species identification of either raw or variously cooked Formosan Reeves’ muntjac meat. It is also possible to apply this method to identify the meat of other wildlife sources.

scholarly journals Microsphere-Based Microfluidic Device for Plasma Separation and Potential Biochemistry Analysis Applications

Micromachines ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 487
Author(s):  
Hongyan Xu ◽  
Zhangying Wu ◽  
Jinan Deng ◽  
Jun Qiu ◽  
Ning Hu ◽  
...  
Keyword(s):  
Blood Sample ◽  
Microfluidic Device ◽  
Biochemical Analysis ◽  
Cost Effective ◽  
Clinical Diagnostics ◽  
Correction Factors ◽  
Separation Device ◽  
Blood Samples ◽  
Plasma Separation ◽  
Blood Biochemical

The development of a simple, portable, and cost-effective plasma separation platform for blood biochemical analysis is of great interest in clinical diagnostics. We represent a plasma separation microfluidic device using microspheres with different sizes as the separation barrier. This plasma separation device, with 18 capillary microchannels, can extract about 3 μL of plasma from a 50 μL blood sample in about 55 min. The effects of evaporation and the microsphere barrier on the plasma biochemical analysis results were studied. Correction factors were applied to compensate for these two effects. The feasibility of the device in plasma biochemical analysis was validated with clinical blood samples.

scholarly journals Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 762
Author(s):  
Yihong He ◽  
Wenxian Chen ◽  
Jindai Fan ◽  
Shuangqi Fan ◽  
Hongxing Ding ◽  
...  
Keyword(s):  
Detection Method ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Porcine Parvovirus ◽  
Swine Industry ◽  
Resource Limited ◽  
Efficient Detection ◽  
Lateral Flow Dipstick ◽  
Polymerase Chain ◽  
Embryonic Death

Porcine parvovirus (PPV) infection is the primary cause of SMEDI (stillbirth; mummification; embryonic death; infertility) syndrome, which is a global burden for the swine industry. Thus, it is crucial to establish a rapid and efficient detection method against PPV infection. In the present work, we developed a recombinase-aided amplification (RAA) assay, coupled with a lateral flow dipstick (LFD), to achieve an amplification of PPV DNA at 37 °C within 15 min. The detection limits of PPV RAA-LFD assay were 102 copies/μL recombinant plasmid pMD19-T-VP1, 6.38 × 10−7 ng/μL PPV DNA, and 10−1 TCID50/mL virus, respectively. This method was highly specific for PPV detection with no cross-reactivity for other swine pathogens. In contrast to polymerase chain reaction (PCR), the PPV RAA-LFD assay is more sensitive and cost-saving. Hence, the established PPV RAA-LFD assay provided an alternative for PPV detection, especially in resource-limited regions.

scholarly journals Evaluation of a lateral flow assay for rapid and simple detection of IFN-γ for the diagnosis of Mycobacterium tuberculosis infection

2020 ◽  
Author(s):  
Jeong-Ran Kim ◽  
Hae Yeong Kang ◽  
Su-Bin Seong ◽  
Nari Kim ◽  
Tae Sun Shim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Tuberculosis Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mycobacterium Tuberculosis Infection ◽  
Blood Samples ◽  
Simple Detection ◽  
Laboratory Workers ◽  
Ifn Γ

Abstract Background: Interferon-gamma (IFN-γ) release assays (IGRAs) are useful for the diagnosis of Mycobacterium tuberculosis infection. Current IGRAs use either enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay, which require complex procedures and techniques to determine IFN-γ secretion. We aimed to compare the usefulness of the easy-to-use lateral flow assay (LFA) with that of the QuantiFERON-TB Gold In-Tube (QFT-GIT) or QuantiFERON-TB Gold Plus (QFT-plus) ELISAs for detecting IFN-γ, produced by the blood T cells stimulated by tuberculosis (TB) antigen. Methods: Following informed consent, 176 participants, including health care workers such as TB laboratory workers and radiologists, were enrolled for the study from June 2017 to June 2018. Blood samples were collected and tested using QFT-GIT and QFT-plus. The secreted IFN-γ was quantified by LFA, which took approximately 15 min, and ELISA, which took approximately 3 h. Results: A total of 176 blood samples were screened. The positive rates of QFT-GIT and QFT-plus were 34.1% and 37.5%, respectively. Overall agreement between QFT-GIT and QFT-plus was 93.1% ( κ = 0.86). The positive rates of LFA with QFT-GIT tube and QFT-plus tube were 25.6% and 31.3%, respectively, overall agreement of LFA being 90.3% ( κ = 0.78) and 89.2% ( κ = 0.77), respectively, compared to the QFT-GIT and QFT-plus ELISA. Conclusion: The ability of LFA to measure IFN-γ was similar to that of ELISA. The current findings suggested that the new LFA could be more conveniently utilized for diagnosing TB infection.

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