Specific Loop-mediated Isothermal Amplification Primers Research Articles

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for Candida glabrata detection.

Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. The specific LAMP primers for C. glabrata detection were designed and evaluated. The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. The developed molecular method would be a promising tool in the management of invasive candidiasis.

Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

Loop-mediated isothermal amplification linked a nanoparticles-based biosensor for detecting Epstein-Barr virus

Epstein-Barr virus (EBV) is a ubiquitous gamma herpesvirus that maintains a lifelong latent association with B lymphocytes. Here, a rapid and reliable diagnosis platform for detecting EBV infection, employing loop-mediated isothermal amplification (LAMP) combined with a gold nanoparticles–based lateral flow biosensors (AuNPs-LFB) (termed LAMP Amplification Mediated AuNPs-LFB Detection, LAMAD), was developed in the current study. A set of specific LAMP primers targeting the Epstein-Barr nuclear antigen (EBNA) leader protein (EBNA-LP) gene was designed and synthesized. Subsequently, these templates extracted from various pathogens and whole blood samples were used to optimize and evaluate the EBV-LAMAD assay. As a result, the limit of detection (LoD) of the EBV-LAMAD assay was 45 copies/reaction. The EBV-LAMAD assay can detect all representative EBV pathogens used in the study, and of note, no cross-reactions were observed with other non-EBV organisms. Moreover, the whole workflow of the EBV-LAMAD assay can be completed within 70 min, including rapid EBV template preparation, EBV-LAMP amplification, and AuNPs-LFB-mediated detection. Taken together, the EBV-LAMAD assay targeting the EBNA-LP gene is a rapid, simplified, sensitive, reliable, and easy-to-use detection protocol that can be used as a competitive potential diagnostic/screening tool for EBV infection in clinical settings, especially in basic laboratories in resource-limited regions.Key points• A novel, simplified, and easy-to-use AuNPs-LFB biosensor was designed and prepared.• LAMP combined with an AuNPs-LFB targeting the novel EBNA-LP gene was established.• EBV-LAMAD is a rapid, sensitive, and reliable detection protocol for EBV infection.Graphical

A LAMP-CRISPR/Cas12b rapid detection platform for canine parvovirus detection.

Canine parvovirus (CPV) is one of the main pathogens causing toxic diarrhea in Chinese dogs, is the cause of large-scale epidemic of dogs, and poses a great threat to the dog industry in China. Rapid, sensitive, and specific CPV testing facilitates the timely diagnosis and treatment of sick dogs. The aim of this study was to build a LAMP-CRISPR/Cas12b platform for CPV detection. The loop mediated isothermal amplification (LAMP) technique was combined with CRISPR-Cas12b analysis to establish a "two-step" and "one-tube" CRISPR/Cas12b rapid CPV method, respectively. The detection system was constructed with specific LAMP primers and single guide RNA (sgRNA) for the highly conserved short fragment of the CPV gene, which could be detected within 1 h without cross-reaction with the other viruses causing canine diarrhea. The detection limits of both "two-step" and "one-tube" CRISPR/Cas12b reactions were 10-1 copies per μL, which was 100 times more sensitive than qPCR and LAMP. In order to achieve point-of-care testing (POCT) of CPV, a one-tube LAMP-CRISPR/Cas12b nucleic acid extraction and detection platform based on magnetic nanoparticle enrichment technology was established to achieve "sample in-result out". The results of this method for simulated samples were compared with those of quantitative real-time PCR; the results showed 100% consistency, and the time was shorter, which could be used to detect the diseased dogs earlier and provide a basis for clinical diagnosis. The LAMP-CRISPR/Cas12b method established in this study provides a sensitive and specific method for rapid detection of CPV, and provides technical support for rapid diagnosis of CPV.

Development of RT-qPCR and loop-mediated isothermal amplification (LAMP) assays for the rapid detection of Rahnella aquatilis in fish

Rahnella aquatilis is widely recognized as an opportunistic pathogen of fish, but molecular detection and diagnostic techniques for it remain to be developed. In a previous study, we isolated the pathogenic R. aquatilis strain KCL-5 from diseased crucian carp Carassius auratus and identified it as a new fish pathogen causing enteritis and septicemia. Here, we describe novel real-time quantitative PCR (RT-qPCR) and loop-mediated isothermal amplification (LAMP) assays for the rapid and sensitive detection of R. aquatilis. Specific RT-qPCR and LAMP primers were developed for the outer membrane protein (OmpA) gene. Their reaction systems were optimized using the recombinant plasmid pMD18-T containing the OmpA gene and their specificities were confirmed using various bacterial fish pathogens including Aeromonas hydrophila, A. veronii, A. allosaccharophila, Yersinia ruckeri, Vibrio vulnificus, V. alfacsensis, Providencia rettgeri, and R. aquatilis. The sensitivities of the RT-qPCR and LAMP assays were further evaluated using serial dilutions of the positive recombinant plasmid pMD18-T/OmpA. Both the RT-qPCR and LAMP assays were able to reliably detect the presence of R. aquatilis with high accuracy and a specificity of 100%. The lowest detection limits of the RT-qPCR and LAMP assays were 2.3 copies/μL and 2.3 × 101 copies/μL, respectively. To the best of our knowledge, this is the first report of RT-qPCR and LAMP methods for R. aquatilis detection, and provides a means for its rapid detection and diagnosis in fish.

Development of a loop-mediated isothermal amplification method for detection of Shewanella algae in fish

Shewanella algae is an opportunistic pathogen, which is widely distributed in freshwater and marine environments. Diseases caused by S. algae have resulted in substantial economic losses in aquaculture industry. This study aimed to establish a loop mediated isothermal amplification (LAMP) method for rapid and specific detection of S. algae in aquatic animals. The specific LAMP primers were designed according to the 16 S rRNA gene sequences of S. algae strains deposited in GenBank. Based on adjustment of the primer concentration and optimization of reaction conditions, the LAMP method for S. algae detection was established, and specificity and sensitivity tests were carried out. The optimal temperature of this method was 65 °C at constant 1 h. The LAMP amplification reaction was positive for S. algae, but negative for Pseudoalteromonas pisicida, Aeromonas hydrophila, A. veronii, Vibrio parahemolyticus, S. decolorationis, S. xiamenensis, Enterobacter cloacae, Bacillus cereus and Staphylococcus epidermidis. The minimum concentration of template DNA of S. algae for the LAMP method was 8.15 × 10−5 µg/µL. The LAMP method was comparable to the common PCR when they were used for detection of S. algae in koi carp artificially infected with this pathogen. In this study, a rapid, specific and sensitive LAMP method was successfully established for the detection of S. algae, providing great convenience for rapid diagnosis of aquatic animal diseases caused by S. algae infection.

Rapid on-site identification of Lepiota brunneoincarnata-induced mushroom poisoning by simple DNA extraction and loop-mediated isothermal amplification strategy

Lepiota brunneoincarnata-induced mushroom poisoning has occurred widely in some areas of the world in recent years, resulting in high mortality and posing potential health risks to humans and pets. However, a rapid and sensitive method for the identification of L. brunneoincarnata is scarce. Loop-mediated isothermal amplification (LAMP) combined with visual dye reaction or lateral flow dipstick (LFD) was established for rapid on-site detection of L. brunneoincarnata. Simple DNA extraction was completed by the heating lysis method within 15 min. In addition, a specific LAMP primer set was designed targeting the internal transcribed spacer (ITS) sequence of L. brunneoincarnata, with no cross-reactivity against 22 other mushroom species. Sensitivity evaluation showed that the detection limits of visual LAMP and LAMP-LFD assays were 10 and 0.1 pg/μL genomic DNA of L. brunneoincarnata, respectively. Moreover, the developed method was successfully applied to cooked mushroom and simulated vomitus, with detection limits of 1% admixture for the visual LAMP assay and 0.1% admixture for the LAMP-LFD assay. The whole detection protocol can be completed in 60 min without expensive instruments, which is suitable for the rapid identification of Lepiota brunneoincarnata-induced mushroom poisoning in the field.

Identification of Gyromitra infula: A Rapid and Visual Method Based on Loop-Mediated Isothermal Amplification.

With mushroom poisoning emerging as one of the most serious food safety problems worldwide, a rapid identification method of poisonous mushrooms is urgently required to investigate the source of poisoning. Gyromitra infula, a kind of poisonous mushroom, contains gyromitrin toxin, which causes epileptogenic neurotoxicity and hemolytic disease. This study aimed to establish a rapid and visual method of G. infula identification based on loop-mediated isothermal amplification (LAMP). A set of specific LAMP primers was designed, and its specificity in G. infula was confirmed against various mushroom species, including its closely related species and other macrofungi. The sensitivity assay showed that the minimum concentration of genomic DNA detected by LAMP was 1 ng/μl. The method’s applicability was conducted by preparing mushroom samples that were boiled and digested in artificial gastric juice. The results showed that the content as low as 1% G. infula can be successfully detected. This method can be completed within 90 min, and the reaction results can be directly observed by the naked eyes. Hence, the identification method of G. infula established based on LAMP in this study is accurate, rapid, sensitive, and low-cost, which is required for clinical treatment or forensic analysis when mushroom poisoning occurs.

Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children.

Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4–100%), specificity 99.0% (95% CI 97.8–99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

A Rapid colorimetric assay for field-portable authentication of Amanita sepiacea using the beta-tubulin gene sequence

Globally, mushroom poisoning is becoming one of the most serious food safety-related problems. To investigate the source of mushroom poisoning, a rapid method for identifying toxic mushrooms is urgently required. This study aimed to establish a colorimetric method based on loop-mediated isothermal amplification (LAMP) for rapid detection of Amanita sepiacea based on the analysis of the beta-tubulin gene. Specific LAMP primers for species specificity identification were designed. The primers designed by the colorimetric method did not cross-react with 41 non-target species mushrooms, and 1 ng of A. sepiacea DNA was detected. The detection method established herein can be used to identify A. sepiacea when present at concentrations as low as 1% in mixed fresh, cooked, or fried mushroom samples. The whole detection process only takes 30 min and requires heating and no complex instrumental help. Therefore, this method can be used in the field detection of poisonous mushrooms.

Rapid and Visual Identification of Chlorophyllum molybdites With Loop-Mediated Isothermal Amplification Method.

Chlorophyllum molybdites is a kind of common poisonous mushroom in China that is widely distributed in different areas. Food poisoning caused by accidentally eating C. molybdites has become more frequent in recent years. In 2019, there were 55 food poisoning incidents caused by eating this mushroom in China. Mushroom poisoning continues to be a common health issue of global concern. When mushroom poisoning occurs, an effective, simple, and rapid detection method is required for accurate clinical treatment or forensic analysis. For the first time, we established a loop-mediated isothermal amplification (LAMP) assay for the visual detection of C. molybdites. A set of specific LAMP primers was designed, and the specificity was confirmed against 43 different mushroom species. The LAMP method could detect as low as 1 pg of genomic DNA. Boiled mushrooms and artificial gastric-digested mushroom samples were prepared to test the applicability of the method, and the results showed that as low as 1% C. molybdites in boiled and digested samples could be successfully detected. The LAMP method can also be completed within 45 min, and the reaction results could be directly observed based on a color change under daylight by the naked eye. Therefore, the LAMP assay established in this study provides an accurate, sensitive, rapid, and low-cost method for the detection of C. molybdites.

A reverse transcription loop‐mediated isothermal amplification assay for the detection of strawberry mottle virus

AbstractStrawberry mottle virus (SMoV) is one of the most important viruses infecting strawberry all over the world, seriously affecting the yield and quality of strawberry especially in the occasion of compound infection. Loop‐mediated isothermal amplification (LAMP) as a new method of nucleic acid amplification has the advantages of rapid detection, simple operation and high sensitivity. Therefore, four specific LAMP primers using the 3′ end conservative coat protein (CP) gene were selected and designed according to the RNA2 sequence of SMoV obtained in NCBI GenBank. Results showed that the optimized RT‐LAMP reaction conditions including 1.0 µmol/L1 primers FIP/BIP, 0.1 µmol/L1 primers F3/B3, 2 mmol/L1 Mg2+, 1.2 mmol/L1 dNTPs, 1.0 mol/L1 Betaine and 2 mol/L1 DTT, with an optimal temperature set at 62°C for 45 min. The amplicons were successfully inspected by visual assessment using SYBR Green I as well as electrophoresis on agarose gel. The sensitivity of the LAMP was 100 times higher than the RT‐PCR method. Furthermore, it was highly specific for SMoV and showed no cross‐reaction with several other strawberry viruses. This assay will provide a practical method for rapid detection of large quantities of field samples and be useful in reducing the virus incidence through production of virus‐free planting material.

Rapid identification of Colchicum autumnale based on loop-mediated isothermal amplification (LAMP) assay

Colchicum autumnale (Colchicaceae) exhibits toxicity, and severe poisoning cases due to ingestion of this plant have been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of the cause of poisoning. In the present study, we developed a rapid and simple method for the detection of C. autumnale based on a loop-mediated isothermal amplification (LAMP) assay, which is 100- to 1000-fold higher than the conventional polymerase chain reaction method. Specific LAMP primers for C. autumnale were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay was performed at an isothermal reaction temperature of 63 ℃. The LAMP reaction was shown to be specific and highly sensitive to C. autumnale, given that the assay can be used for 10 pg of purified DNA. Using a simple protocol for on-site detection, the entire procedure from pretreatment to evaluation required approximately 1 h. Moreover, the LAMP method using a microfluidic device detected multiple genes, including C. autumnale-specific DNA regions, at a time. The currently proposed protocol exhibits good potential as a screening method for C. autumnale poisoning in emergency medical care.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

BackgroundThis study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.ResultsA set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.ConclusionsThe analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Detection of Vibrio Pathogens from Cultured Turbot (Scophthalmus maximus) by Loop-Mediated Isothermal Amplification (LAMP)

Abstract Vibrio species are the major concern to the aquaculture industry because they are both fish pathogens and also bring foodborne diseases to human. Loop-mediated isothermal amplification (LAMP) is a simple and rapid method to detect the pathogens. In this study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Vibrio scophthalmi, a pathogen of turbot. 16s rDNA of Vibrio scophthalmi was used as the template to design a set of specific LAMP primers. The LAMP primers were designed, according to our requirements, by Beijing Lanpu Bio-tech Co. Ltd. using Primer Explorer V4 software (http://primerexplorer.jp/e/v4_manual/index.html). The DNA of intestinal pathogens of turbot was extracted using proteinase K kit (Beyotime). By initial trial at 64 ℃ of water bath the reaction become visible in turbid or in fluorescent color after 20 min. Three different turbot fish were positive, indicating that they were containing Vibrio scophthalmi in their intestine and this signifies that all the primers bind specifically to the gene of 16S rRNA gene, in another word they are pathogen positive. To confirm the specific amplification of the 16S rRNA gene, gel electrophoresis was run. The resulted bands were specific to the region of 16SrRNA gene supposed to be amplified. The lowest detection limit of the LAMP assay was the DNA concentration of 1.25 × 10-17 μg/ml. This LAMP method can be of high value to the food industry due to its various advantages such as speed, specificity, sensitivity, cost- and labor-efficiency.

Simple and visible detection of duck hepatitis B virus in ducks and geese using loop-mediated isothermal amplification

In this study, loop-mediated isothermal amplification (LAMP) was used to establish a rapid, specific, and visual detection method for duck hepatitis B virus (DHBV). The design and synthesis of 4 specific LAMP primers were based on the conserved gene region of the DHBV genome, and the optimum temperature and time of the LAMP reaction were 63°C and 50 min, respectively. The LAMP assay was confirmed to be specific for DHBV detection and had the same sensitivity as the quantitative PCR assay. A visual detection method for rapid determination of results was developed using a color indicator containing phenol red and cresol red. A color change was produced based on a pH change in the reaction system, indicating a positive reaction. For the detection of samples from ducks and geese, the LAMP method has the advantages of simplicity, high sensitivity and specificity, good visibility, and low cost. Moreover, it is more practical and convenient than PCR-related assays for the clinical detection of DHBV.

Development and Evaluation of Isothermal Amplification Methods for Rapid Detection of Lethal Amanita Species.

In the present work, loop-mediated isothermal amplification (LAMP) and hyperbranched rolling circle amplification (HRCA) methods were developed to detect and distinguish different lethal Amanita species. Specific LAMP primers and HRCA padlock probes for species-specific identification and a set of universal LAMP primers for lethal Amanita species were designed and tested. The results indicated that the LAMP-based assay was able to discriminate introclade lethal Amanita species but was not able to discriminate intraclade species perfectly, while the HRCA-based assay could discriminate whether introclade or intraclade species. The universal LAMP primers were positive for 10 lethal species of Amanita section Phalloideae and negative for 16 species of Amanita outside section Phalloideae. The detection limits of LMAP and HRCA were 10 and 1 pg of genomic DNA per reaction, respectively. In conclusion, the two methods could be rapid, specific, sensitive and low-cost tools for the identification of lethal Amanita species.

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification.

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

Species-specific detection of medically important aspergilli by a loop-mediated isothermal amplification method in chronic pulmonary aspergillosis.

Chronic pulmonary aspergillosis (CPA) is a common subtype of pulmonary aspergillosis and a life-threatening disease. However, its diagnosis remains difficult due to the lack of specific clinical features and radiologic findings, as well as the difficulty of isolating Aspergillus spp. We developed a novel species-specific detection method of medically important aspergilli using a loop-mediated isothermal amplification (LAMP) for CPA. Specific LAMP primer sets for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans were designed. The use of the LAMP assay was validated using respiratory specimens (CPA cases, n = 21; nonaspergillosis cases, n = 23). A total of 15 cases were positive in the CPA group (A. fumigatus, n = 5; A. flavus, n = 1; A. niger, n = 1; A. terreus, n = 7; A. nidulans, n = 1), but only three in the non-CPA group (A. niger, n = 2; A. terreus n = 1). The sensitivity and specificity of the diagnosis of CPA by the LAMP system were 71.4% and 87.0%, respectively. In conclusion, we developed a species-specific detection approach for five medically important aspergilli using the LAMP method. The system showed high sensitivity and specificity for diagnosis of CPA.