scholarly journals Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chika Takano ◽  
Yoko Kuramochi ◽  
Mitsuko Seki ◽  
Dong Wook Kim ◽  
Daisuke Omagari ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Detection Limits ◽  
Public Health Problem ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Identification ◽  
Loop Mediated Isothermal Amplification ◽  
Pcr Assays ◽  
Blood Specimens

AbstractIn children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). However, since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. However, invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. To develop a rapid, simple, and cost-effective assay to detect serotypes of Streptococcus pneumoniae, we developed a novel loop-mediated isothermal amplification (LAMP) assay based on the sequences available for the 13 capsular types that are included in PCV13: 1, 3, 4, 5, 6 A, 6B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, and 23 F. We evaluated test reactivity, specificity, sensitivity and performance, and compared the results between established LAMP and conventional PCR assays. To support its clinical use, the detection limits of the LAMP assay were evaluated using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) and blood specimens. We confirmed the specificity of the LAMP assay using 41 serotypes of pneumococcal strains. The sensitivity of the LAMP assay was 10 to 100 copies per reaction, compared to 10 to 104 copies per reaction for PCR assays. The detection limits of the LAMP assay were comparable when using DNA-spiked CSF and blood specimens, as compared to using purified DNA as the template. In conclusion, a rapid and simple LAMP-based pneumococcal serotyping method has been developed. This is the first report of a LAMP method for a PCV13 serotype-specific identification assay, which could be a promising step to facilitate epidemiological studies of pneumococcal serotyping.

scholarly journals 23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246699
Author(s):  
Jiwon Lee ◽  
Youngbae Yoon ◽  
Eun Jin Kim ◽  
Donghyun Lee ◽  
Yeongjun Baek ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Limit Of Detection ◽  
Public Health Problem ◽  
Lamp Assay ◽  
Polysaccharide Vaccine ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Pcr Assays ◽  
Blood Specimens

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

scholarly journals Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

2011 ◽  
Vol 47 (No. 4) ◽  
pp. 140-148 ◽  
Author(s):  
N. Rostamkhani ◽  
A. Haghnazari ◽  
M. Tohidfar ◽  
A. Moradi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Transgenic Cotton ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Leaf Tissues

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T<sub>2</sub> transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65&deg;C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10<sup>&ndash;1</sup> to 10<sup>&ndash;8</sup>) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

scholarly journals Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

2008 ◽  
Vol 57 (4) ◽  
pp. 439-443 ◽  
Author(s):  
Basu Dev Pandey ◽  
Ajay Poudel ◽  
Tomoko Yoda ◽  
Aki Tamaru ◽  
Naozumi Oda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Rrna Gene ◽  
Predictive Values ◽  
Loop Mediated Isothermal Amplification ◽  
System A

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5–97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Rapid Detection of Mycoplasma pneumoniae by Loop-Mediated Isothermal Amplification (LAMP) in Clinical Respiratory Specimens

2019 ◽  
Author(s):  
Maryam ARFAATABAR ◽  
Narjes NOORI GOODARZI ◽  
Davoud AFSHAR ◽  
Hamed MEMARIANI ◽  
Ghasem AZIMI ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Respiratory Specimens

  Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clini-cal specimens collected from patients with pneumonia. Methods: Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined. Results: Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /μL or ~ 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was ‘substantial’ (κ=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the con-gruence between LAMP assay and PCR assay was ‘almost perfect’ (κ=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR. Conclusion: Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid, Sensitive Detection of Campylobacter jejuni in Cattle Farm Samples

2014 ◽  
Vol 77 (9) ◽  
pp. 1593-1598 ◽  
Author(s):  
HEE-JIN DONG ◽  
AE-RI CHO ◽  
TAE-WOOK HAHN ◽  
SEONGBEOM CHO
Keyword(s):  
Campylobacter Jejuni ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Cattle Farm ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Pcr Assays ◽  
Template Dna ◽  
Commercial Kit

Campylobacter jejuni is a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuni in naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipO gene. The LAMP assay was specific (100%inclusivity and exclusivity for 84 C. jejuni and 41 non–C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R2 = 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejuni strains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipO gene was tested using the LAMP assay. Amplification of the hipO gene by LAMP within 30 min (mean =10.8 min) in all C. jejuni isolates (n = 51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuni was compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuni in cattle farm samples, may play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the risk of human campylobacteriosis.

scholarly journals Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation

2012 ◽  
Vol 79 (1) ◽  
Author(s):  
Jian-min Zhang ◽  
Hai-yan Shen ◽  
Ming Liao ◽  
Tao Ren ◽  
Li-li Guo ◽  
...  
Keyword(s):  
16S Rrna ◽  
South China ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Lamp Method ◽  
Haemophilus Parasuis ◽  
Loop Mediated Isothermal Amplification ◽  
Glässer’S Disease ◽  
Glasser's Disease

Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal amplification (LAMP) test was developed to improve the specificity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly amplified the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classified into 9 serovars and had 37 genetic patterns when analysed by pulsed-field gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, specificity and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer’s disease.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Assay to De-tect Toxoplasmosis in Schizophrenia Patients

2020 ◽  
Author(s):  
Hadi MIRAHMADI ◽  
Raheleh HASANZADEH ◽  
Hamid MALEK RAEESI ◽  
Shirzad FALLAHI ◽  
Mahdi KHOSHSIMA SHAHRAKI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Efficiency ◽  
Parasitic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Intermediate Hosts ◽  
Cross Sectional ◽  
Loop Mediated Isothermal Amplification ◽  
Schizophrenic Patients

Background: Toxoplasma gondii (T. gondii) causes an important parasitic infection known as toxoplasmosis, which is a globally distributed important zoonosis. One of the major serious characteristics of T. gondii is its ability to manipulate the behavior of intermediate hosts. We performed a cross-sectional study to determine toxoplasmosis in schizophrenic patients, as one of the major neuropsychiatric disorders, using loop-mediated isothermal amplification (LAMP) technic by targeting parasite B1 gene. Methods: Blood samples were taken from 118 schizophrenic patients hospitalized in tow hospitals including Baharan, Clinic of Psychiatric Ali-ibn-Abi-Talib Hospital (in Zahedan City), and Amir-al Momenin Psychiatric Hospital (in Zabol City), Sistan and Baluchestan Province, southeast Iran in 2016. They were analyzed using LAMP, and compared with the previous data of nested-PCR and serology. Results: Out of the 118 schizophrenic individuals, 56 patients (47.4%) were found to be infected with T. gondii. The diagnosis of toxoplasmosis was confirmed in 41 patients (34.7%) via the nested-PCR. The seroprevalence of toxoplasmosis in schizophrenic patients was 55.9% (66/118). Conclusion: We found a high efficiency of LAMP method in identifying toxoplasmosis and its high prevalence among schizophrenic patients. Our findings could provide viable offer implications for the prevention of schizophrenia.

scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Salmonella spp. in Terms of Sensitivity and Applicability

2020 ◽  
pp. 1-5
Author(s):  
Yanhong Liu ◽  
Deguo Wang ◽  
Meng Zhang ◽  
Yanhong Liu ◽  
Yongzhen Wang
Keyword(s):  
Genomic Dna ◽  
Pathogenic Bacteria ◽  
Detection Limits ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
23S Rrna ◽  
Lamp Method ◽  
Salmonella Spp ◽  
Loop Mediated Isothermal Amplification ◽  
Food Borne

Salmonella spp. are important food-borne pathogens that can cause diseases in humans. Many detection methods have been established in Salmonella spp. using loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). The detection limits of these assays varied from 1 CFU/reaction to 104 CFU/reaction, from 100 fg genomic DNA/reaction to 10 pg genomic DNA/reaction, or from 2.0×101 CFU/mL to 107 CFU/mL for food samples. In this study, LAMP assays were developed using genomic DNA for the detection of Salmonella spp. Two sets of LAMP primers were designed using the invA gene and the 16S-23S rRNA intergenic spacer region (ITS) of S. enterica as the target sequences for two LAMP assays. The detection limits of the two methods were respectively 20 pg S. enterica DNA/reaction and 10 pg S. enterica DNA/reaction at the optimized temperature, and the LAMP methods were of high repeatability and specificity for S. enterica detection. This study provides a baseline for the application of LAMP for the detection of food-borne pathogenic bacteria.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of viable but non-culturable Vibrio cholerae O1

2021 ◽  
Author(s):  
Parastoo Chamanrokh ◽  
Rita R. Colwell ◽  
Anwar Huq
Keyword(s):  
Vibrio Cholerae ◽  
Culture Media ◽  
Lamp Assay ◽  
Environmental Water ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Waterborne Pathogen ◽  
Loop Mediated Isothermal Amplification ◽  
Added Benefit ◽  
Polymerase Chain

<i>Vibrio cholerae</i>, an important waterborne pathogen, is a rod-shaped bacterium that naturally exists in aquatic environments. When conditions are unfavorable for growth, the bacterium can undergo morphological and physiological changes to assume a coccoid morphology. This stage in its life cycle is referred to as viable but non-culturable (VBNC) since VBNC cells do not grow on conventional bacteriological culture media. The current study compared polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to detect and identify VBNC <i>V. cholerae</i>. Because it is difficult to detect and identify VBNC <i>V. cholerae</i>, the results of the current study are useful in showing LAMP to be more sensitive and rapid than PCR in detecting and identifying non-culturable, coccoid forms of <i>V. cholerae</i>. Furthermore, the LAMP method is effective in detecting and identifying very low numbers of coccoid VBNC <i>V. cholerae</i> in environmental water samples, with the added benefit of being inexpensive to perform.

scholarly journals Development of Loop-Mediated Isothermal Amplification for Detection ofLeifsonia xylisubsp.xyliin Sugarcane

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Jing Liu ◽  
Liping Xu ◽  
Jinlong Guo ◽  
Rukai Chen ◽  
Michael Paul Grisham ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Time Saving ◽  
Visual Judgment ◽  
Coryneform Bacterium ◽  
Loop Mediated Isothermal Amplification ◽  
Total Dna ◽  
Sensitive Method

Ratoon stunt, caused by the xylem-limited coryneform bacteriumLeifsonia xylisubsp.xyli(Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detectingLxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification ofLxxand without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused byLxxin sugarcane. This is the first report of LAMP-based assay for the detection ofLxxin sugarcane.

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