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Results for “Isothermal amplification”

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PubMed2003

Loop-mediated isothermal amplification for detection of African trypanosomes.

While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.

Animals↗

PubMed2005

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions.

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.

Adult↗

PubMed2005

Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.

Animals↗

PubMed2003

Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction.

The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of amplification of TYLCV genomic DNA without gel electrophoresis.

Amino Acid Sequence↗

PubMed2004

Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method.

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future.

Animals↗

PubMed2005

Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP).

Annual seasonal outbreaks of respiratory syncytial virus (RSV) infection occur every winter. Most patients are diagnosed clinically by a rapid detection kit for RSV protein(s) from nasopharyngeal secretion (NPS), but some problems have been reported on the specificity and sensitivity of such rapid detection kits. To ratify these issues, a sensitive, specific, simple, and rapid molecular based diagnostic method is expected to be introduced and we have developed a method to detect the RSV genome of subgroups A and B independently by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We detected the genomic RNA corresponding approximately to 0.1 TCID 50 in the sample by RT-LAMP for both RSV subgroups under isothermal condition within 60 min after extraction of RNA. Specific DNA amplification was monitored by a real-time turbidimeter and the quantity of RNA was calculated. The RSV genome was detected in 47 of 50 NPS by RT-LAMP, and in 42 by nested RT-PCR, whereas virus isolation was positive for 29 and enzyme-linked immunoassay (EIA) for 34. RSV subgroup A was detected in 25 by RSV RT-LAMP A, RSV subgroup B in 23 by RSV RT-LAMP B, and dual infection with RSV subgroups A and B was identified in one case. They were confirmed with digestion with a specific restriction enzyme, Bgl II. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RSV with high sensitivity similar to nested RT-PCR.

Genome, Viral↗

PubMed2004

Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method.

Detection and analysis of Babesia gibsoni infection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs had B. gibsoni DNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to published B. gibsoni sequences. The limits of detection of B. gibsoni parasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection of B. gibsoni infections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection of B. gibsoni infections in dogs.

Animals↗

PubMed2005

An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification.

BACKGROUND: Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment. RESULTS: A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV. CONCLUSION: This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

Animals↗

PubMed2006

Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10(-3) FFU of the cell-culture propagated viruses. The reaction time needed to detect 10(4) FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa.

Animals↗

PubMed2007

Rapid detection of metastasis of gastric cancer using reverse transcription loop-mediated isothermal amplification.

Tailor-made surgeries for patients with solid malignancies have been under consideration on the basis of the development of new approaches for minor metastatic foci of malignant tumors. Accurate and reliable methods to detect metastases in biopsy specimens with certain rapidity are essential for the performance of these surgeries. The aim of this study was to develop a rapid and practical method to detect metastasis in specimens from patients with gastric carcinoma with the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction, a novel technique for detecting mRNA expressions of targeted sequences with high sensitivity, specificity and rapidity under isothermal conditions. RT-LAMP primers to detect cytokeratin19 (CK19) mRNA were generated and 92 lymph nodes (LNs) obtained from 9 patients with gastric cancer were tested for tumor metastases with this technique. Among 92 LNs, 15 were metastasis-positive by routine histopathological examination. RT-LAMP reaction detected CK19 expression in all of the pathologically positive LNs and in 16 of 77 negative LNs. Nested RT-PCR assay for CK19 expression was also performed on 2 of the 9 cases including 32 LNs. The agreement rate of CK19 expression detection by RT-LAMP and RT-PCR analysis was 31/32 (97%). The RT-LAMP technique showed similar sensitivity to detect metastases as nested RT-PCR assay, with a rapidity comparable to that of intraoperative histopathological examination with frozen sectioning and hematoxylin and eosin staining. This method is expected to play an essential role in the performance of tailor-made surgeries in the near future.

Aged↗

PubMed2005

Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method.

Ochroconis gallopava is a species of dematiaceous fungi recognized as a causative agent of zoonotic and emerging fungal infections. It affects the central nervous system and respiratory tracts of humans, birds and cats. We designed O. gallopava species-specific primer sets to aid in its identification by a loop-mediated isothermal amplification (LAMP) method based on the D1/D2 domain of the LSU rDNA sequence. The LAMP method successfully detected the gene from both fungal DNA and experimentally infected brains and spleens of mice and will be helpful in the diagnosis of O. gallopava infection.

Animals↗

PubMed2005

A simple method for the detection of measles virus genome by loop-mediated isothermal amplification (LAMP).

Approximately 20,000-30,000 measles patients were reported in a surveillance of infectious diseases because of low vaccine coverage of 80% in Japan. Among them, some were thought to be secondary vaccine failure (SVF) with generally mild or non-typical measles illness and sometimes became a source of further transmission. We have developed a new, sensitive, and rapid method to detect the measles virus genome by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We examined 50 nasopharyngeal secretion (NPS) samples that were obtained during the 1999 outbreak and stored at -70 degrees C and fresh NPS, lymphocytes and sera from 11 patients in 2003. Total RNA was extracted from the samples and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and RT-LAMP. We detected the genomic RNA corresponding to at least 0.01-0.04 TCID50, 30-100 copies in samples by RT-LAMP within 60 min after extraction of RNA, and all four genotypes isolated in Japan were equally amplified. Specific DNA amplification was monitored spectrophotometrically by real time turbidimeter and the quantity of RNA was calculated. Measles virus genome was detected in 44 of 50 stored NPS by RT-PCR and in 49 by RT-LAMP. The vaccine strain was discriminated from wild strains after sequencing the LAMP products. RT-LAMP is a useful rapid diagnostic method for the detection of measles virus without any special apparatus, showing higher sensitivity than RT-PCR, and expected to be applied for hospital-based infection control and for laboratory-based measles surveillance.

Base Sequence↗

PubMed2006

Simple differentiation method of mumps Hoshino vaccine strain from wild strains by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

Mumps virus is still circulating and annual mumps outbreaks occur with fluctuating magnitudes in Japan. Aseptic meningitis has been reported after vaccination and it would be of importance to determine whether this was related to the vaccination. The objective of this study was to develop a sensitive, specific and rapid diagnostic method for the differentiation of the Hoshino vaccine strain from circulating wild types. We developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method of the hemagglutinin neuraminidase (HN) region for the detection of mumps virus genome from clinical samples. The typical ladder pattern disappeared after the LAMP products of the Hoshino vaccine strain were digested with ScaI, but those of wild types were not cut by ScaI. We obtained 19 cerebro spinal fluids (CSF) from the patients with aseptic meningitis and 17 salivary swab samples from the patients with acute parotitis after mumps vaccination, in which one case was complicated with orchitis. Mumps virus genome was detected in 18 CSF samples and in all NPS by RT-LAMP. The Hoshino vaccine strain was identified in 16 out of 18 CSF RT-LAMP positives and in 11 out of 17 NPS samples and the remaining samples were identified as wild types. RT-LAMP followed by ScaI digestion is a sensitive, simple and rapid differential method and useful for laboratory surveillance for vaccine-adverse events.

Base Sequence↗

PubMed2005

Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification.

BACKGROUND: Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. METHODS: Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. RESULTS: The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. CONCLUSIONS: Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.

Animals↗

PubMed2005

Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification.

Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.

Animals↗

PubMed2006

Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori.

Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori, detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non-H. pylori bacterial species, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Helicobacter hepaticus, and 51 H. pylori strains. Results showed that H. pylori-specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 10(2) CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection.

Base Sequence↗

PubMed2005

Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe-based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop-mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP-based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture-associated pathogens is discussed.

Animals↗

PubMed2001

Isothermal amplification and multimerization of DNA by Bst DNA polymerase.

We have demonstrated the isothermal in vitro amplification and multimerization of several different linear DNA targets using only two primers and the strongly strand-displacing exonuclease-negative Bst DNA polymerase. This reaction has been termed linear target isothermal multimerization and amplification (LIMA). LIMA has been compared with cascade rolling-circle amplification and has been found to be less sensitive but to yield similar variable-length multimeric dsDNA molecules. Products from several different LIMA reactions were characterized by restriction analysis and partial sequence determination. They were found to be multimers of subsets of the target sequence and were not purely primer derived. The sensitivities with respect to target concentration of several different LIMA reactions were determined, and they varied from 0.01 amol to 1 fmol. The sensitivity and specificity of LIMA were further tested using E. coli genomic DNA, and the selective amplification of a transposon fragment was demonstrated. A successful strategy for reducing LIMA-dependent background DNA synthesis in rolling-circle amplification embodiments was devised. This entailed the affinity purification of circular DNA templates before amplification.

Base Sequence↗