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At least 37 records · Page 2Linked to original sources
PubMed2006

Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP).

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The whole procedure is very simple and rapid, and reaction time and temperatures were optimized for 60 min at 65 degrees C, respectively. Detection of gene amplification could be accomplished by agarose gel electrophoresis. The standardized RT-LAMP procedure was used to detect YHV in the heart and gill from infected shrimp. Thus, the RT-LAMP assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for YHV detection in shrimp.

Base Sequence↗
PubMed2002

Accelerated reaction by loop-mediated isothermal amplification using loop primers.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using additional primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of analysis including detection is less than 1h, this new method should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.

Base Sequence↗
PubMed2004

Analysis of specific gene by integration of isothermal amplification and electrophoresis on poly(methyl methacrylate) microchips.

We have successfully achieved the integration of isothermal amplification and the subsequent analysis of specific gene fragments on poly(methyl methacrylate) microchips. In our experiments, loop-mediated isothermal amplification, which can offer higher specificity and efficiency than PCR, has been performed at a constant temperature (65 degrees C). After amplification, products could be either examined by the integrated microchip-based electrophoresis or directly observed by naked eye with SYBR Green I added into the reaction solution. By such an integrated microsystem, the amplification and the subsequent analysis of prostate-specific antigen gene with template concentration at 23 fg/microL could be finished within 15 min, which demonstrates its advantages of high specificity, good reproducibility, and fast speed in gene detection.

DNA↗
PubMed1995

Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) irrespective of the stage of HIV-1 infection.

Using an experimental assay for isothermal amplification of HIV RNA in plasma (NASBA, Organon Teknika, Boxtel, The Netherlands), 70 samples from 59 HIV-1-infected persons and 29 samples from 28 uninfected volunteer blood donors were tested for the presence of HIV-1 RNA. The HIV-1-positive plasma samples were drawn from patients at various stages of infection: two samples from persons with signs of acute HIV infection (CDC stage I); 29 samples from 25 symptom-free persons (CDC stage II) and 39 samples from 32 persons with AIDS-related symptoms (CDC stage IV). All samples from HIV-1-infected persons had HIV-1 RNA, irrespective of the stage of infection (100% sensitivity). Testing the donor samples in duplicate, initially 54/58 samples (93%) tested negative for HIV-1 RNA. Repeated testing of the 4 samples with inconsistent test results showed all samples to be negative (specificity 100%). Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) promises to be a valuable alternative to the detection of HIV-1 RNA or DNA by the polymerase chain reaction.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Rapid detection of Epstein-Barr virus DNA by loop-mediated isothermal amplification method.

BACKGROUND: The loop-mediated isothermal amplification (LAMP) method is a novel technique for the amplification of specific DNA sequences. OBJECTIVES: To establish the LAMP method for amplifying Epstein-Barr virus (EBV) DNA and to examine its reliability for the detection of EBV DNA in clinical specimens. STUDY DESIGN: Sera from 108 patients, who were initially suspected of primary EBV infection, were tested by the EBV LAMP method, and the results were compared with those of the real-time PCR assay. Serological examination was regarded as the standard diagnostic method. RESULTS: To diagnose primary EBV infection, the sensitivity of LAMP was 86.4% and the specificity was 100%. The sensitivity of the real-time PCR assay was 84.1% and the specificity was 98.4%. Longitudinal analysis showed that the detection rate of EBV DNA in serum by the LAMP method decreased with time in accordance with the decrease of the EBV load. EBV DNA could not be detected in serum 40 days after onset of symptoms. CONCLUSIONS: These results indicate that the sensitivity and specificity of the LAMP method are comparable to those of real-time PCR and that detecting EBV DNA in serum by this method is potentially useful for diagnosing primary EBV infection.

Child, Preschool↗
PubMed2007

Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method.

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.

Adolescent↗
PubMed2004

Detection of human herpesvirus 7 DNA by loop-mediated isothermal amplification.

The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a sensitivity of 500 and 250 copies/tube for 30- and 60-min reactions, respectively. Following these initial validation studies, clinical samples collected from two patients with primary HHV-7 infections were examined by HHV-7 LAMP. By use of agarose gel electrophoresis, HHV-7 LAMP products could be detected in acute-phase plasma samples but no LAMP product was detectable in convalescent-phase plasma samples from either patient. Since a turbidity assay is less sensitive than agarose gel electrophoresis, no HHV-7 LAMP product could be detected in plasma samples after a 30-min LAMP reaction. After a 60-min LAMP reaction, HHV-7 LAMP product could be detected in acute-phase plasma samples.

Base Sequence↗
PubMed2005

Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification.

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus.

Bacterial Proteins↗
PubMed2006

Sensitive and rapid diagnosis of human parvovirus B19 infection by loop-mediated isothermal amplification.

BACKGROUND: Human parvovirus B19 (B19) infections exhibit various skin manifestations that are similar to and hence hard to distinguish from many other skin diseases. The virological diagnosis of B19 infection is usually based on time-consuming serological tests and polymerase chain reaction (PCR). OBJECTIVES: In this study, a DNA amplification method, loop-mediated isothermal amplification (LAMP), was used for the diagnosis of B19 infection and was compared with PCR. METHODS: Ten patients with acute B19 infection and 16 patients with other skin diseases were enrolled. Sera and pharyngeal swabs were used directly as the templates in LAMP. The LAMP reaction was carried out at 63 degrees C for 1 h in a heat block. The reaction products were judged visually, by adding SYBR Green I into the tubes, and by gel electrophoresis. RESULTS: B19 DNA was detected by LAMP in 10 sera and all of seven tested pharyngeal swabs of 10 patients with acute B19 infection but not in samples from 16 patients with other skin diseases. The results were in agreement with those obtained by PCR except for one case. The reason for the single discrepancy may be that the sensitivity of LAMP is 10(2) times higher than PCR. CONCLUSIONS: Detection of B19 DNA by LAMP in serum and especially in the pharynx is a rapid and convenient method for the diagnosis of acute B19 infection.

Acute Disease↗
PubMed1997

Monitoring the amplification of CATCH, a 3SR based cooperatively coupled isothermal amplification system, by fluorimetric methods.

Three different types of fluorescence detection methods were employed to monitor amplification of a previously established isothermal cooperatively coupled amplification system as it can serve as a tool for the investigation of fundamental issues in evolutionary optimization. By using 5'IRD-41 fluorescent labeled primers, the intercalating dye TOPRO-1 and a 5'fluorescin/3'DABCYL 4-(4-dimethylamino-phenylazo)benzoic acid labeled ss 24 nt DNA, evolving molecular cooperation is accessible, sequence specifically as well as non-sequence-specifically without using radioactivity.

DNA↗
PubMed2004

Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP) method.

PURPOSE: To develop a new method to detect and type adenoviruses directly from conjunctival scrapings using loop-mediated isothermal amplification (LAMP) with adenovirus (ad) type specific primer. METHODS: Using primers specific for the gene of ad1, ad3, ad4, ad8, ad19 and ad37, heat denatured adenovirus DNA was amplified by the LAMP and polymerase chain reaction (PCR). Alkaline lysed adenovirus prototype and conjunctival scrapings were also used directly as templates. RESULTS: Type specific primers amplified ad genes of the corresponding ad prototype specifically. The specific amplification was observed in both heat denatured and alkaline lysed samples. The amplified product was first detected within 45 min. Ad genotypes of clinical samples determined by the LAMP method were almost identical to those determined using the PCR-sequencing method. CONCLUSIONS: LAMP based isothermal amplification of adenovirus genome for detection and typing of adenoviruses is faster than PCR based methods. This new method will be useful for rapid diagnosis and typing of adenoviral conjunctivitis.

Adenovirus Infections, Human↗
PubMedDate not supplied

Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP) method.

PURPOSE: To develop a new method to detect and type adenoviruses directly from conjunctival scrapings using loop-mediated isothermal amplification (LAMP) with adenovirus (ad) type specific primer. METHODS: Using primers specific for the gene of ad1, ad3, ad4, ad8, ad19 and ad37, heat denatured adenovirus DNA was amplified by the LAMP and polymerase chain reaction (PCR). Alkaline lysed adenovirus prototype and conjunctival scrapings were also used directly as templates. RESULTS: Type specific primers amplified ad genes of the corresponding ad prototype specifically. The specific amplification was observed in both heat denatured and alkaline lysed samples. The amplified product was first detected within 45 min. Ad genotypes of clinical samples determined by the LAMP method were almost identical to those determined using the PCR-sequencing method. CONCLUSIONS: LAMP based isothermal amplification of adenovirus genome for detection and typing of adenoviruses is faster than PCR based methods. This new method will be useful for rapid diagnosis and typing of adenoviral conjunctivitis.

Adenovirus Infections, Human↗
PubMed2005

Rapid detection of herpes simplex virus DNA in cerebrospinal fluid: comparison between loop-mediated isothermal amplification and real-time PCR.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69 cerebrospinal fluid (CSF) samples from patients suspected of having HSV infection of the central nervous system. The results of the real-time PCR analysis and nested PCR assay were in complete accord. When nested PCR was regarded as standard, the sensitivity of LAMP was 81%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 90%. Although further improvement is necessary for the wide spread use, the LAMP method might be applicable to diagnosis of HSV infection of the central nervous system.

Adolescent↗
PubMed2006

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification.

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.

Bacterial Toxins↗
PubMed2006

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method.

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.

Animals↗
PubMed2005

Detection of Salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates.

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37 degrees C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37 degrees C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably.

Animals↗