Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria

Diagnosis of malaria is a prominent challenge due to the endemic nature of infection. Malaria poses a great threat to global public health. The disease can be diagnosed by several techniques out of which microscopy is a known gold standard. High sensitivity of molecular techniques is making them more reliable and popular as tools for diagnosis of malaria. However, new methods are required which can fulfill the criteria of being Point of Care Test (POCT) as defined by WHO. Loop-mediated isothermal amplification (LAMP) technique amplifies DNA in an isothermal condition, and surpasses the disadvantages of conventional molecular techniques such as polymerase chain reaction. Multiplex LAMP, a modification of LAMP may emerge as a new POC for malaria diagnosis. This review deals with the use of LAMP and multiplex LAMP in diagnosis of malaria and its prospective use as point of care techniques.

Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus / 生物工程学报

Three pairs of primers were designed according to the conserved region of IBRV gB gene published in GenBank(GenBank Accession No. DQ006857.1) using the software Primer Explorer V4. The loop mediated isothermal amplification (LAMP) assay was established by optimization of the reaction system and then evaluated through sensitivity and specificity tests. In total 393 clinical specimens were detected for IBRV using the established LAMP assay performed at 65℃ for 50 min, which produced a ladder-like pattern of amplification bands and the detection result could be judged by color change. The sensitivity of the assay was 10 copies/μL plasmid DNA which was 1000 times higher than that by PCR method and equivalent to nested-PCR. There was no cross-reactivity of the assay with bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and vesicular stomatitis virus (VSV). The positive rate of 301 nasal swabs and 92 serum specimens were 87.6% and 58.8%, respectively, which meant nasal swab specimen was more suitable for clinical IBRV detection by the method. The IBRV LAMP method established in this study has the advantages of visualization, quickness, specificity and sensitivity and be suitable for rapid detection of clinical IBRV detection on the spot.

Comparision on the effect of RT-LAMP and LAMP method for detection of Mycobacterium tuberculosis / 中国人兽共患病学报

We compared the RT-LAMP,LAMP and L-J for detecting MTB to provide the rapid diagnosis method for tu-berculosis.In this assay,NTM and other common respiratory bacteria were used to detect specificity,Mycobacterium tubercu-losis specific products were identified by restriction enzyme digestion.To detect sensitivity,we used RT-LAMP,LAMP and L-J to detect 100 cases sputum specimens from patients with tuberculosis and 22 cases control sputum specimens,the 10 times dilution of 1 ng/μL H37Rv standard strains was used to detect RT-LAMP limit.The results showed that the positive rate of RT-LAMP,LAMP and L-J were 100%,92%,88%.RT-LAMP and L-J,RT-LAMP and LAMP were statistically signifi-cant,RT-LAMP had 10 times higher sensitivity than LAMP,RT-LAMP not only to identify viable but also capable of detec-ting a single copy of MTB.So RT-LAMP is superior to LAMP and L-J and is practical for use in primary medical care institu-tion or peripheral laboratory.

Rapid Detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring blaVIM-2, blaIMP-1 and blaOXA-23 Genes by Using Loop-Mediated Isothermal Amplification Methods

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla OXA-23, which are critical components for carbapenem resistance. METHODS: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO4) by testing different concentrations of MgSO4. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates. RESULTS: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles. CONCLUSIONS: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.

Molecular Diagnosis of Streptococcus pneumoniae in Middle Ear Fluids from Children with Otitis Media with Effusion

PURPOSE: The long-term administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) of young children with otitis media with effusion (OME). The purpose of this study is to determine whether molecular diagnostics can be used for rapid and direct detection of the bacterial pathogen in culture-negative MEFs. METHODS: The specificity and sensitivity of both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to the lytA gene of Streptococcus pneumoniae were comparatively tested and then applied for pneumococcal detection in the clinical MEFs. RESULTS: The detection limit of the PCR assay was approximately 10(4) colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of S. pneumoniae. Both PCR and LAMP did not amplify nucleic acid at over 10(6) CFU of H. influenzae or M. catarrhalis, both of which were irrelevant bacterial species. Of 22 culture-negative MEFs from children with OME, LAMP positivity was found in twelve MEFs (54.5%, 12/22), only three of which were PCR-positive (25%, 3/12). Our results showed that the ability of LAMP to detect pneumococcal DNA is over four times higher than that of PCR (P<0.01). CONCLUSIONS: As a high-resolution tool able to detect nucleic acid levels equivalent to <10 CFU of S. pneumoniae in MEFs without any cross-reaction with other pathogens, lytA-specific LAMP may be applied for diagnosing pneumococcus infection in OME as well as evaluating the impact of a pneumococcal conjugate vaccine against OME.

Rapid detection of Salmonella Typhi by loop-mediated isothermal amplification (LAMP) method

An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.

Loop-mediated isothermal amplification assays for screening of bacterial integrons

BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

Detection of Acute Toxoplasmosis in Pigs Using Loop-Mediated Isothermal Amplification and Quantitative PCR

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/microl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63degrees C by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1alpha) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Identification and detection of Cordyceps sinensis with LAMP / 中草药

Objective: The method of loop-mediated isothermal amplification (LAMP) was employed to detect and identify Cordyceps sinensis rapidly. Methods: Specific LAMP primers were designed according to the CS2 serine protease (csp2) gene of Cordyceps sinensis. C. sinensis DNA was extracted using CTAB method. The reaction conditions of LAMP were optimized. Specificity of LAMP reaction was validated by six different strains and using restriction enzyme Taq1 digested the LAMP products. Sensitivity of LAMP was tested with diluted C. sinensis solution with 10-fold gradient. LAMP products were shown by gel electrophoresis or adding SYBR Green I. Results: The method of LAMP for detecting C. sinensis was effective and specific. The detection limit of LAMP assay was up to 6 pg/mL. Conclusion: LAMP protocol is a promising method for the identification and detection of C. sinensis and Chinese materia medica as well.

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2 / 中国病毒学·英文版

In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.

Detection of Plasmodium vivax sporozoites-carrying mosquitoes using loop-mediated isothermal amplification (LAMP) / 中国血吸虫病防治杂志

Objective To establish a simple,convenient,quick and high sensitive method of loop-mediated isothermal amplification (LAMP) for detection of Plasmodium vivax-carrying mosquitoes.Methods The species conservative regions of P.v CSP gene were selected to design 2 pairs of primers which recognized 6 distinct regions.To evaluate the specificity of detection by LAMP,infected Anopheles,An.sinensis (An.s),Plasmodium falciparum (P.f),and healthy human blood DNA were selected as templates.To assess the sensitivity of detection,1.3×10~6,1.3×10~5,1.3×10~4,1.3×10~3,1.3×10~2,1.3×10~1 and 1.3×10~0 copies of P.v CSP plasmid DNA mixed with 1.0 μl An.s DNA were used as the templates of LAMP.The infected An.s DNAs were diluted with negative An.s DNA by 1:2,1:4,1:8,1:16,1:32,1:64,1:128 and 1:256 and then detected by LAMP to show the sensitivity of batch quantity detection.The applied value of this method was evaluated by detecting the same batch of 67 artificial infected An.s mosquitoes,and compared with the detection of microscopic examination and nested PCR in parallel.Results By using LAMP,the detection of infected An.s was positive,while the control samples were all negative.The limits of detection of different proportion dilutions of the mixture of P.v CSP plasmid DNA with An.s DNA were 1.3×10~2 copies.The limits of detection of different proportion dilutions of the mixture of infected An.s DNA with An.s DNA were 1:128.The positive rate of detecting the same batch of 67 artificial infected mosquitoes was 47.76% by LAMP,25.37% by the microscopic examination (X~2 = 7.24,P0.05).Compared with the test of the microscopic examination and then with a statistical analysis,the sensitivity of LAMP was 100%,which agreed well with the sensitivity of nested PCR (100%).Conclusion The method of LAMP is simple,convenient and high sensitive,and it is a potential method for detecting Plasmodium vivax-carrying mosquitoes in the field.

One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA / 中国病毒学·英文版

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

Development of Loop-mediated Isothermal Amplification (LAMP) Method for Detection of Vibrio parahaemolyticus / 中国生物工程杂志

Vibrio parahaemolyticus has been considered as one of the most important foodborne bacterial pathogens.The loop-mediated isothermal amplification(LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of this pathogen for the first time.A set of four primers,two outer and two inner primers,was designed specifically to recognize the thermolabile hemolysin gene(tlh) of V.parahaemolyticus.The LAMP reaction mix was optimized.The most optimal reaction temperature and time of the LAMP assay for the tlh gene were 60℃ and 60min,respectively.Genomic DNAs from 28 bacterial strains including 14 V.parahaemolyticus strains were amplified using LAMP,and no amplicon was observed in other bacterial strains.The detection limit of this LAMP assay was around 90 fg of V.parahaemolyticus genomic DNA and 24 colonies forming units for pure cultures.In addition,this method was applied to detect artificially contaminated food samples,and the detection limit was 89 cfu/g for non-cultured artificially contaminated food samples.These results suggested that detection of V.parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment.This assay is expected to become a valuable tool for rapid detection and identification of V.parahaemolyticus.