Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Nie, Kai; Qi, Shunxiang; Zhang, Yong; Luo, Le; Xie, Ying; Yang, Mengjie; Zhang, Yi; Jin, Li; Shen, Hongwei; Li, Qi; Ma, Xuejun

doi:10.1371/journal.pone.0052486

Cited by 48 publications

(46 citation statements)

References 27 publications

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“…The only limitation of RT-LAMP is the extraction of RNA under field conditions. However, Fukuta et al (17) and Nie et al (18) reported that RT-LAMP can be performed on crude samples without RNA extraction. The crude samples were not tested by RT-LAMP in this study, but this could be a better option and it may replace the other diagnostic tests in the field.…”

Section: Discussionmentioning

confidence: 99%

One-step real-time loop-mediated isothermal amplification (RT-LAMP): evaluation and its application for the detection of foot-and-mouth-disease virus and its serotypes

Maryam¹,

Rasheed²,

Latif³

et al. 2017

Turk J Vet Anim Sci

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Availability of a rapid and sensitive diagnostic technique is key in successful prevention and control of infectious diseases like foot-and-mouth disease (FMD). Existing conventional diagnostic tests for FMD are laborious and time-consuming with low sensitivity and specificity. Molecular-based techniques are costly and difficult, involving refined apparatus like a thermal cycler. In the present study, the technique of real-time loop-mediated isothermal amplification (RT-LAMP) was standardized for diagnosing FMDV and its serotypes, evaluated using field samples, and compared with the existing real-time PCR in Pakistan. RT-LAMP amplified the target 3D gene using specific primers at 65 °C for 60 min and the VP1 gene using serotype specific primers at 63 °C for 60 min. A total of 38 samples out of 50 were positive by RT-LAMP and identified serotypes were A (n = 15), O (n = 15), and Asia-1 (n = 8). The efficiency of RT-LAMP in this study was highest for serotype Asia-1 (80.9%) followed by serotype A (73.4%) and O (62.35).

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Section: Discussionmentioning

confidence: 99%

One-step real-time loop-mediated isothermal amplification (RT-LAMP): evaluation and its application for the detection of foot-and-mouth-disease virus and its serotypes

Maryam¹,

Rasheed²,

Latif³

et al. 2017

Turk J Vet Anim Sci

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“…GQ279371.1) from the GenBank database (http: //www.ncbi.nlm.nih.gov/) were used to design the primers for the EV71 and CVA16 RT-IMSA assays, respectively. These sequence regions selected were highly conserved regions of the EV71 and CVA16 VP1 genes, which were used and evaluated previously in our laboratory for the primer designs for the RT-LAMP assay (4,18,23). The primer design for the RT-IMSA assay was mainly based on the rules for that of the RT-LAMP assay, with the aid of PrimerExplorer V4 (see http://primerexplorer.jp /elamp4.0.0/; Eiken, Kyoto, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4)(5)(6). Nucleic acid amplification techniques, such as reverse transcription-PCR (RT-PCR) (7)(8)(9) and reverse transcription-quantitative PCR (qRT-PCR) (10, 11) take less time (2 to 3 h) and are generally used for making a standard diagnosis due to a high detection limit.…”

mentioning

confidence: 99%

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

et al. 2014

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c Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-footand-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-selfmatching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R 2 values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R 2 values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses. Hand-foot-and-mouth disease (HFMD) is a childhood syndrome and is characterized by tiny blisters on the skin and oral mucosa together with fever and poor appetite. The two major causative agents of HFMD are human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). EV71-related HFMD commonly accounts for the fatal cases, due to severe complications, such as brainstem encephalitis and rapid fatal pulmonary edema, whereas CVA16-related HFMD usually presents with mild symptoms (1-3). Since no vaccine or antiviral drugs are currently available to treat HFMD, the early and rapid detection of EV71 and CVA16 are critical for the prevention and control of HFMD infection.Presently, laboratory detection of EV71 and CVA16 includes traditional virus isolation, neutralization, and nucleic acid amplification techniques. It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4-6). Nucleic acid a...

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“…In addition, it has been reported that the sensitivity of molecular detection methods such as qPCR and LAMP is subject to a variety of factors present in clinical samples. For example, the multitude of drugs and fluids prescribed to patients at risk for IA can inhibit the qPCRs (36), while in comparison, the Bst polymerase in LAMP is more tolerant to these inhibitors than Taq polymerase in the amplification of the clinical samples (37), which might lead to better sensitivity for the LAMP assay.…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

et al. 2016

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Aspergillus fumigatus is a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection of A. fumigatus infection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection of A. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatus strains, including 5 species of the Aspergillus genus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10 2 copies for the same target. Clinical samples from a total of 69 patients with probable IA (n ‫؍‬ 14) and possible IA (n ‫؍‬ 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection of A. fumigatus in clinical testing has been developed.A spergillus fumigatus, a ubiquitous saprophytic fungus in most parts of the world, is a conditional pathogenic fungus and the major cause of invasive aspergillosis (IA). The infection is initiated by inhalation of conidia, which are cleared quickly in a normal host but can cause invasive disease in immunocompromised individuals (1). Despite many efforts during the last decade, the morbidity and mortality due to IA has been increasing (2). The poor therapeutic outcomes are associated with the weak host immune status and, importantly, the delayed detection and identification of the causative agent (3).The traditional culturing method, which is the gold standard for the diagnosis of A. fumigatus, requires several days for observation of growth, is time-consuming, and has only moderate specificity. Laboratory methods for the diagnosis of A. fumigatus include mainly direct microscopic examination, histopathological studies, and serum galactomannan (GM) and (1¡3)-␤-D-glucan assays. The measurement of galactomannan in bronchoalveolar lavage (BAL) fluid shows promise for enhancing the diagnosis of A. fumigatus (4-7), but only approximately 50% of BAL fluid measurement results can match the results of culture and direct examination (8).PCR-based molecular detection systems for fast and sensitive identification by examination of small amounts of DNA in serum and blood have been developed (9-11). However, the lack of standardization for critical activities in the methods and the evaluation of clinical samples has hampered the development of PCR-based diagnostic tests for clinical use....

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Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Cited by 48 publications

References 27 publications

One-step real-time loop-mediated isothermal amplification (RT-LAMP): evaluation and its application for the detection of foot-and-mouth-disease virus and its serotypes

One-step real-time loop-mediated isothermal amplification (RT-LAMP): evaluation and its application for the detection of foot-and-mouth-disease virus and its serotypes

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

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