Reverse Transcriptase Loop-mediated Isothermal Amplification Research Articles

Development of a Reverse Transcriptase Loop-Mediated Isothermal Amplification (LAMP) Assay for the Sensitive Detection of Leishmania Parasites in Clinical Samples

Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% (N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% (N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.

Optimisation of reverse transcriptase loop-mediated isothermal amplification assay for rapid detection of Macrobrachium rosenbergii noda virus and extra small virus in Macrobrachium rosenbergii

The standardisation and optimisation of a one step single tube reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii noda virus ( MrNV) and extra small virus (XSV), in giant fresh water prawn, M. rosenbergii. Time, temperature and quantity of each reagent were optimised for the detection of the two viruses. This method was more sensitive than the conventional reverse transcriptase polymerase chain reaction (RT-PCR) for detecting the two viruses. The RT-LAMP reaction is highly suited for disease diagnosis in developing countries. Amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of a whitish precipitate of magnesium pyrophosphate as a by-product. The cost of RT-LAMP for one reaction is nearly 4 times less than that of RT-PCR.

Rapid and specific detection of H3 swine influenza virus using reverse transcription loop-mediated isothermal amplification method

The main objective of our study is to develop a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based system for rapid and specific detection of H3 swine influenza virus (SIV). The system, H3 RT-LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT-LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10-fold more sensitive than the conventional RT-PCR assay and even comparable to the real-time RT-PCR method, with the detection limit of about one plaque-forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT-LAMP and real-time RT-PCR methods, while only 7 were positive with the conventional RT-PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT-PCR and real-time RT-PCR approaches. Our results provide the first direct evidence that RT-LAMP is highly specific and sensitive for detecting H3 SIV. These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.

Rapid Detection of Matrix Metalloproteinase-7 and Carcinoembryonic Antigen mRNA Expression in Intraoperative Pleural Lavage Samples Using the Reverse Transcriptase Loop-Mediated Isothermal Amplification Method

To detect the RNA of the tumor origin in intraoperative pleural lavage accurately by examining using rapid nucleic acid amplification method because patients with lung cancer often have recurrence detected by a cytologic examination of cancer cells in intraoperative pleural lavage.We used reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), which enables rapid gene amplification, and examined the expression of matrix metalloproteinase-7 (MMP-7) and carcinoembryonic antigen (CEA) mRNA in intraoperative pleural lavage samples obtained from 79 consecutive surgical patients with lung adenocarcinoma.MMP-7 mRNA was detected in cancer tissues from 76 (96%) of those cases, while it was also detected in the intraoperative pleural lavage samples of 23 (30%) of those 76 cases. In addition, CEA mRNA was detected in cancer tissues from 55 (70%) cases and in 8 (15%) of the lavage samples. MMP-7 mRNA-positive cases showed a significantly positive correlation with the p and n factors, and moderate to poor differentiation. Three cases shown to be positive in cytology examinations were also positive for both MMP-7 and CEA mRNA by RT-LAMP.This detection method can be utilized during an operation and may provide useful information.

A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus

This report describes the development of a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the detection of swine vesicular disease virus (SVDV). The assay detects the virus rapidly, within 30–60 min and the result is visualised either by gel-electrophoresis or by the naked eye through the addition of SybrGreen. A collection of 28 SVDV isolates were tested positive, while heterologous viruses such as foot-and-mouth disease virus and vesicular stomatitis virus remained negative. The performance of the RT-LAMP was compared directly with real-time PCR using RNA from clinical samples including nasal swabs, serum and faeces. For nasal swabs and serum the sensitivity of the RT-LAMP was shown to be at least equivalent to real-time PCR. Interestingly, for faecal samples the RT-LAMP assay was shown to be even more sensitive than real-time PCR, possibly because it is less sensitive to inhibitory substances. This RT-LAMP assay provides a number of benefits for the diagnosis of SVD, since the assay is sensitive and rapid, and the isothermal amplification strategy used is not reliant upon expensive equipment it is particularly suited for “front line” diagnosis of SVD in modestly equipped laboratories, in field stations or in mobile diagnostic units.

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 °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.

Development of H5-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection

We developed a rapid and sensitive diagnosis system for H5N1 highly pathogenic avian influenza (HPAI) virus infection using an unique gene amplification method, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). The sensitivity of the system was found to be 100-fold higher than that of ordinary one-step RT-PCR. Moreover, by using viral RNAs extracted from influenza viruses of all 15 HA subtypes, the RT-LAMP system was confirmed to amplify only the RNA of H5 subtype virus. In the surveillance of H5N1 virus infection of wild birds, we detected two positive cases from dead crows found near the affected area with H5N1-HPAI by using RT-LAMP system, although one of two positive cases was missed by RT-PCR. These results suggested that our newly developed RT-LAMP system specific for H5 virus would be a beneficial diagnostic tool for surveillance of recent outbreaks caused by H5N1-HPAI viruses.

Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: Comparison with immunochromatography and virus isolation

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was established for the detection of influenza A virus H1 and H3 subtype strains and influenza B virus strains specifically. The total procedure from RNA extraction to virus typing was completed within 3 h. In terms of specificity, the representative AH1, AH3 and B strains were detected only by strain-specific primers respectively. No cross-detection was observed. In terms of sensitivity, virus was detected at a minimum concentration of 10 ffu/ml. Eighty-three nasopharyngeal aspirates obtained from children diagnosed clinically with influenza were tested by the RT-LAMP assay, along with commercially available immunochromatography rapid diagnostic tests and by virus isolation. Virus was isolated from 78 samples (94%) and the subtype was determined by the hemagglutination inhibition test. Although it took at least 3 days, the detection sensitivity was the best of the three methods. With two rapid assays, the detection sensitivity of the RT-LAMP assay (85.5%) was higher than that of immunochromatography tests (75.9%). In addition, the RT-LAMP assay can be used to differentiate emerging influenza virus subtypes by selecting appropriate primer sets.