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Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV. Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection. Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.
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Objective To investigate the effects of reverse transcription loop-mediated isothermal amplifica-tion(RT-LAMP)method and RT-PCR method for detecting Mycobacterium tuberculosis(MTB)in cerebro-spinal fluid(CSF)to provide a basis for its rapid diagnosis and clinical pharmacodynamic evaluation.Methods Eighty-five cases of CSF sample in the Bethune International Peace Hospital of PLA from December 2015 to April 2017 were selected for conducting the study and divided into the tuberculous meningitis(TBM)group(46 cases),suspected TBM group(25 cases)and control group(16 cases).The 16S rRNA region of MTB was used to design the specific primers.Then RT-LAMP and RT-PCR detection technological systems were estab-lished.Then the detection results by using the these two methods was analyzed.Results The positive detec-tion rates of the TBM group were 97.8% and 75.0% respectively,which of the suspected TBM group were 76.0% and 40.0% respectively,and which of the control group were 0.0% and 12.0% respectively,the posi-tive detection rate of each group in the RT-LAMP method was higher than that in the RT-PCR method,the difference was statistically significant(P<0.01);in the control group,adopting RT-PCR detection found non-specific amplification,while which was not found by adopting RT-LAMP method,indicating that the specifici-ty of RT-LAMP method was stronger than that of RT-PCR;the sensitivity of RT-PCR was 10.0 CFU/mL, which was higher than 1 CFU /mL of RT-LAMP.Conclusion RT-LAMP has the advantages of simpleness,sensitivity,rapidness and detecting viable bacteria,compared with PCR,which has strong specificity,easy op-erating,low cost and short time-consuming,is expected to be a routine detection tool of basic level and field medical institutions and developing countries.
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We compared the RT-LAMP,LAMP and L-J for detecting MTB to provide the rapid diagnosis method for tu-berculosis.In this assay,NTM and other common respiratory bacteria were used to detect specificity,Mycobacterium tubercu-losis specific products were identified by restriction enzyme digestion.To detect sensitivity,we used RT-LAMP,LAMP and L-J to detect 100 cases sputum specimens from patients with tuberculosis and 22 cases control sputum specimens,the 10 times dilution of 1 ng/μL H37Rv standard strains was used to detect RT-LAMP limit.The results showed that the positive rate of RT-LAMP,LAMP and L-J were 100%,92%,88%.RT-LAMP and L-J,RT-LAMP and LAMP were statistically signifi-cant,RT-LAMP had 10 times higher sensitivity than LAMP,RT-LAMP not only to identify viable but also capable of detec-ting a single copy of MTB.So RT-LAMP is superior to LAMP and L-J and is practical for use in primary medical care institu-tion or peripheral laboratory.
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Objective To detect influenza A virus by reverse transcription‐loop mediated isothermal amplification (RT‐LAMP) assay to established a rapid ,simple and visualization nucleic acid detection method .Methods The RT‐LAMP primers were designed in accordance with the hemagglutinin gene of influenza A virus .Then ,the specificity of the primers was evaluated by detection of different influenza viruses ,and the sensitivity was confirmed by testing multiple diluted RNA samples . Hydroxynaphthol blue (HNB) was used for visually evaluation and gel electrophoresis was used for validation .Clinical samples were detected by RT‐LAMP assay .Its consistency with fluorescent quantitative polymerase chain reaction (PCR) methods was compared .Results The primers of RT‐LAMP assay had high specificity . This technique could amplify influenza A virus accurately .The detection limit of RT‐LAMP assay was 2 .5 × 103 copies/mL by detection of multiple diluted RNA samples .In addition ,the results of RT‐LAM P assay could be visually inspected using HNB by color change ,and the results was in accordance with that of gel electrophoresis . RT‐LAMP assay was in consistence with fluorescent quantitative PCR when clinically applied .Conclusions RT‐LAMP assay is a rapid ,specific ,sensitive and simple method to detect influenza A virus .
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Objective To verify the reliability of our previously established reverse-transcription loop-mediated isother-mal amplification ( RT-LAMP) method for the detection of sentinel lymph nodes metastasis in breast cancer patients .Meth-ods Sentinel lymph nodes of breast cancer patients were analyzed by RT-LAMP and FDA-approved GeneSearch methods respectively, and the consistency of the two methods was assessed with a kappa concordance test.Results One hundred and thirty-four cases of sentinel lymph node samples were collected from seven hospitals in Beijing .Using the GeneSearch assay as the gold standard, the sensitivity, specificity and consistentcy of RT-LAMP were 96.2%(25/26),96.3%(104/108) and 96.3%(129/134), respectively.Statistical analysis showed that the two methods were consistent (Kappa=0.8857, P<0.001).Conclusion RT-LAMP is highly consistent with GeneSearch ,efficient,simple and inexpensive, promising a good prospect of application to intra-operative detection of sentinel lymph nodes metastasis for breast cancer patients.
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Purpose: The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop‑mediated isothermal amplification (LAMP) technique. Materials and Methods: A reverse‑transcription loop‑mediated isothermal amplification (RT‑LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT‑LAMP was tested, and the clinical specimens was assayed by the RT‑LAMP comparing with conventional reverse‑transcription polymerase chain reaction (RT‑PCR) and real‑time PCR. Results: A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT‑LAMP. The detection rate for EV71 was 56.89% by RT‑LAMP, 41.38% by real‑time PCR and 34.48% by RT‑PCR. The minimum detection limit of RT‑LAMP was 0.01 PFU, both of RT‑PCR and real‑time PCR was 0.1PFU. Non‑cross‑reactive amplification with other enteroviruses was detected in the survey reports. Conclusions: The effectiveness of RT‑LAMP is higher than RT‑PCR and real‑time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource‑poor countries or in developing countries.
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A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.
Subject(s)
Animals , Foot-and-Mouth Disease/diagnosis , Foot-and-Mouth Disease Virus/genetics , Nucleic Acid Amplification Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Reverse Transcription/genetics , Sensitivity and SpecificityABSTRACT
Reverse transcription loop-mediated isothermal amplification(RT-LAMP)is a novel nucleic acid amplification technique which has been mainly used in a variety of viruses and other pathogens of gene detection.Compared with reverse transcriptase polymerase chain reaction (RT-PCR),the RT-LAMP has advantages of reaction simplicity,rapidly,cost-effective,detection sensitivity and specificity.Nowadays,RT-LAMP technology is gradually used in various medical fields,but it is still a new method especially in cancer diagnosis of tumor gene.The article reviews the application of the RT-LAMP technology in the cancer
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Purpose: The objective of this study was to establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of human immunodeficiency virus type 1 (HIV-1). Materials and Methods: The HIV-1 integrase gene region was selected because it was a conserved part of the HIV-1 genome. Six primers specific to eight regions of the HIV-1 integrase gene were designed. A total of 171 samples (18 HIV-1 confirmed positive samples and 153 serum specimens were collected in this study) were tested by RT-LAMP and reverse-transcription polymerase chain reaction (RT-PCR). After amplification in an isothermal water bath for 45 min, samples containing HIV-1 generated the expected ladder-like products while other viruses generated no product. Results: The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with RT-PCR. The assay was significantly more sensitive than normal gel-based RT-PCR. Conclusion: Because it is specific and simple, the RT-LAMP assay can be widely applied in clinical laboratories for rapid detection of HIV-1.
Subject(s)
HIV-1/analysis , HIV-1/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , RNA, Viral/analysis , RNA, Viral/genetics , Sensitivity and SpecificityABSTRACT
Background: Coxsackie B viruses (genus, Enterovirus; family, Picornaviridae) can cause aseptic meningitis, encephalitis, pleurodynia, and fatal myocarditis, and are implicated in the pathogenesis of dilated cardiomyopathy. The differentiation of the group B Coxsackieviruses into their subtypes has potential clinical and epidemiological implications. Objective: In this study, we developed a one-step, single-tube genogroup-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of group B Coxsackie genomes targeting 5′ UTR region. Materials and Methods: The amplification can be obtained in less than 1 hour by incubating all the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis and the monitoring of gene amplification can also be visualised with the naked eye by using SYBR green I fluorescent dye. Results: A total of 40 samples comprising 31 positive samples and 9 negative samples were used in this study for comparative evaluation. The results were compared with those from Real-Time Polymerase Chain Reaction (RT-PCR). None of the RT-PCR-positive samples were missed by RT-LAMP, thereby indicating a higher sensitivity of the RT-LAMP assay. Conclusion: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid detection of non-polio enterovirus (NPEV) not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.
Subject(s)
Clinical Laboratory Techniques/economics , Clinical Laboratory Techniques/methods , Coxsackievirus Infections/diagnosis , Electrophoresis, Agar Gel , Enterovirus B, Human/genetics , Enterovirus B, Human/isolation & purification , Humans , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Organic Chemicals/metabolism , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Staining and Labeling/methods , Temperature , Time FactorsABSTRACT
A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic,prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV)strains.As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR),the RT-LAMP method only used a turbidimeter,exhibited a detection limit corresponding to a 10-4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV containing cells,and no cross-reactivity was observed with other related viruses including porcine circovirus type 2,swine influenza virus,porcine rotavirus and classical swine fever virus.From forty-two field samples,33 samples in the RT-LAMP assay was detected positive,whereas three of which were not detected by RT-PCR.Furthermore,in 33 strains of PRRSV,an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates,especially in developing countries.
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A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV.PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.
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Objective To use a new simple Reverse Transcription Loop-mediated isothermal amplification(RTLAMP) method was applied to detect rubella virus nucleic acid and compared with Reverse TranscriptionPolymerase Chain Reaction (RT-PCR).Method Comparing the detection rate of the RT-LAMP method with that of RT-PCR for detecting rubella virus nucleic acid from rubella virus.Results The nucleic acid positive rates of all 11 strains of rubella virus were 100% by the two methods,the positive rate was 55%.Conclusion RT-LAMP is more simple and convenient than RT-PCR.