scholarly journals Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 897
Author(s):  
Lavel Chinyama Moonga ◽  
Kyoko Hayashida ◽  
Naoko Kawai ◽  
Ryo Nakao ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Detection System ◽  
Spotted Fever ◽  
Simultaneous Detection ◽  
Febrile Illness ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Spotted Fever Group ◽  
Loop Mediated Isothermal Amplification ◽  
Rickettsia Monacensis ◽  
Sfg Rickettsia

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis’s clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

scholarly journals Establishment of a Rapid Detection Method for Rice Blast Fungus Based on One-Step Loop-Mediated Isothermal Amplification (LAMP)

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 1967-1973 ◽  
Author(s):  
Ling Li ◽  
Shu Ya Zhang ◽  
Chuan-Qing Zhang
Keyword(s):  
Rapid Detection ◽  
Rice Blast ◽  
Detection System ◽  
Fungal Spores ◽  
Lamp Assay ◽  
Rice Blast Fungus ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Blast Fungus

Rice blast is one of the most serious diseases for rice, and controlling the filamentous fungus Magnaporthe oryzae that causes rice blast is crucial for global food security. Typically, early infected rice does not show symptoms. Therefore, the early diagnosis of rice blast is particularly important to avoid uncontrollable propagation of rice blast fungus. In the present work, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect the pathogen at the early infected stage of rice. The Alb1 superfamily hypothetical protein MGG_04322, a nuclear shuttling factor involved in ribosome and melanin biogenesis, was chosen as the target for designing the LAMP primers. The LAMP assay enabled rapid detection of as little as 10 pg of pure genomic DNA of M. oryzae. In addition, we established the quantitative LAMP (q-LAMP) detection system to quantify the conidia of rice blast fungus. The q-LAMP assay enabled rapid detection (within 35 min) of the fungal spores at a sensitivity of 3.2 spores/ml. In addition, the assay sets up the linearization formula of the standard curve as y = 0.3066 + 15.33x (where x = amplification of time), inferring that spore number = 100.60y. In addition, the q-LAMP assay was successfully used to detect the presence of the virulence strains of M. oryzae (wild type) in comparison with that of the two mutant strains by quantifying the biomass within host tissue. These results provide a useful and convenient tool for detecting M. oryzae that could be applied in the incubation period of rice blast before symptoms appear.

scholarly journals Molecular Investigation of Tick-Borne Pathogens in Ticks removed from Tick-Bitten Humans in the Republic of Korea

2019 ◽  
Author(s):  
Mi-Seon Bang ◽  
Choon-Mee Kim ◽  
Sang-Hyun Pyun ◽  
Dong-Min Kim ◽  
Na Ra Yun
Keyword(s):  
Tick Species ◽  
Spotted Fever ◽  
Common Species ◽  
Republic Of Korea ◽  
Spotted Fever Group ◽  
Haemaphysalis Longicornis ◽  
Babesia Gibsoni ◽  
Specific Pcr ◽  
Rickettsia Monacensis ◽  
Sfg Rickettsia

Abstract Background Tick-borne infections are continuously increasing due to climate change, increased outdoor activities and increased travel between countries. This study was to investigate the presence of tick-borne pathogens in ticks removed from tick-bitten humans in southwest provinces of Republic of Korea (ROK). Methods Ticks were obtained from those tick-bitten humans between May 2014 and September 2017 in Jeollanam provinces and Gwangju metropolitan city in ROK. The presence of the tick-borne pathogens in ticks removed from tick-bitten humans was analyzed using pathogen-specific polymerase chain reaction (PCR). Results We identified 33 ticks from three tick species, namely Amblyomma testudinarium (60.6%), Haemaphysalis longicornis (27.3%), and Ixodes nipponensis (12.1%) in order of occurrence by morphology and 16S rDNA-targeting PCR. Tick-borne pathogens were found in 16 ticks using pathogen-specific PCR. From the results, 12 ticks (36.4%) tested positive for spotted fever group (SFG) Rickettsia: Rickettsia monacensis (1/12), R. tamurae (8/12), and Candidatus Rickettsia jingxinensis (3/12). Three ticks (9.1%) were positive for Anaplasma phagocytophilum . In addition, three ticks (9.1%) tested positive for Babesia gibsoni (1/3) and B. microti (2/3). Conclusions In conclusion, we identified three tick species; the most common species was A. testudinarium followed by H. longicornis and I. nipponensis . SFG Rickettsia , A. phagocytophilum , and Babesia spp. were the most frequently detected pathogens in ticks removed from tick-bitten humans. R. tamurae and Ca. R. jingxinensis were firstly detected in Korea. The present results will contribute to the understanding of tick-borne infections in animals and humans in the ROK.

scholarly journals Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

2020 ◽  
Vol 21 (22) ◽  
pp. 8741
Author(s):  
Güven Edgü ◽  
Lena Julie Freund ◽  
Stefanie Hartje ◽  
Eckhard Tacke ◽  
Hans-Reinhard Hofferbert ◽  
...  
Keyword(s):  
Disease Management ◽  
Management Practices ◽  
Tobacco Rattle Virus ◽  
Simultaneous Detection ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Tuber Quality ◽  
Loop Mediated Isothermal Amplification ◽  
Tuber Necrosis ◽  
Corky Ringspot

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

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

2004 ◽  
Vol 28 (6) ◽  
pp. 445-450 ◽  
Author(s):  
Taketoshi Wakabayashi ◽  
Ryoko Yamashita ◽  
Tetsuhiko Kakita ◽  
Mito Kakita ◽  
Tetsuro Oshika
Keyword(s):  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification

scholarly journals Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

Sign in / Sign up

Export Citation Format

Share Document