Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care

Abstract Background C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for “naked eye” end-point detection when testing ‘real-world’ clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. Results The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91–100%. Conclusions This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing ‘real-world’ samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

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Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽

10.1371/journal.pone.0244753 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0244753

Author(s):

Jeeyong Kim ◽

Borae G. Park ◽

Da Hye Lim ◽

Woong Sik Jang ◽

Jeonghun Nam ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Positive Reaction ◽

Real Time Pcr ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Published Data ◽

Analytical Sensitivity ◽

Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

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EFFECTIVENESS OF THE LOOP-MEDIATED ISOTHERMAL AMPLIFICATION WITH FLUORESCENT DETECTION IN THE DIAGNOSIS OF PARVOVIRUS ENTERITIS IN CARNIVORES

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2019-1-90-95 ◽

2019 ◽

Vol 1 (1) ◽

pp. 90-95 ◽

Cited By ~ 1

Author(s):

O. A. Petrusha ◽

T. L. Chernichenko ◽

I. A. Kofiadi ◽

V. V. Zverev ◽

E. B. Faizuloev

Keyword(s):

Real Time ◽

Point Of Care ◽

Reference Method ◽

Diagnostic Value ◽

Isothermal Amplification ◽

Fluorescent Detection ◽

Lamp Method ◽

Analytical Sensitivity ◽

Loop Mediated Isothermal Amplification ◽

Syto 9

The aim of the study was to evaluate the diagnostic value of the method of loop-mediated isothermal amplification of DNA with real-time fluorescent detection (real-time LAMP, or RT-LAMP) on the model of carnivore parvoviruses. Materials and methods. Samples of feces, blood and swabs from the rectum of different species of predatory animals with parvovirus enteritis (n = 39) and healthy animals (n = 31), as well as laboratory strains of mink enteritis virus, were analyzed by RT-LAMP using SYTO-9 and SYTO-82 dyes. Real-time PCR was used as a reference method. Results. In our study, the LAMP method with real-time fluorescence detection (RT-LAMP) in the carnivore parvovirus enteritis model provides high analytical sensitivity (1.5×103 copies of DNA/ml), diagnostic sensitivity and specificity (up to 100% under optimal conditions). Comparison of the two intercalating dyes showed that the SYTO-82 dye provides a higher signal-to-background ratio (22.6 ± 2.1) than the SYTO-9 dye (6.3 ± 1.5) (p <0.0000001 ). At the same time, SYTO-9 dye at the sensitivity limit (10 copies of DNA) provides an increase in fluorescence in the reaction mixture 13 minutes earlier than for SYTO-82 (23 and 36 minutes, respectively). Conclusion. RT-LAMP is a promising method for rapid and highly sensitive «point-of-care» diagnosis of infectious diseases, as well as in conditions of livestock farms or in field conditions.

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Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens:Chlamydia psittaciandChlamydia pecorum

PeerJ ◽

10.7717/peerj.3799 ◽

2017 ◽

Vol 5 ◽

pp. e3799 ◽

Cited By ~ 26

Author(s):

Martina Jelocnik ◽

Md. Mominul Islam ◽

Danielle Madden ◽

Cheryl Jenkins ◽

James Branley ◽

...

Keyword(s):

Real Time ◽

Pathogen Detection ◽

Point Of Care ◽

Chlamydia Psittaci ◽

Isothermal Amplification ◽

Diagnostic Tools ◽

Clinical Samples ◽

Conserved Gene ◽

Thermal Cycler ◽

Species Specific

BackgroundChlamydia psittaciandChlamydia pecorumare important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robustC. psittaci-specific andC. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens.Methods and MaterialsThe LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of theC. psittaci-specific Cps_0607 gene or a 209 bp region of aC. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity forC. psittaciandC. pecorumLAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples,C. psittaciLAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while forC. pecorumLAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36C. pecorumsamples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays.ResultsBoth LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min forC. psittaciandC. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% forC. psittaci(91.7% sensitivity and 92.9% specificity); and 84.1% forC. pecorum(90.6% sensitivity and 77.4% specificity). For a subset of 36C. pecorumsamples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP.DiscussionIn this study, we describe the development of novel, rapid and robustC. psittaci-specific andC. pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.

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Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

Molecular Medicine ◽

10.1186/s10020-021-00289-0 ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Bruna de Oliveira Coelho ◽

Heloisa Bruna Soligo Sanchuki ◽

Dalila Luciola Zanette ◽

Jeanine Marie Nardin ◽

Hugo Manuel Paz Morales ◽

...

Keyword(s):

Viral Load ◽

Internal Control ◽

Reverse Transcription ◽

Color Change ◽

Point Of Care ◽

Colorimetric Detection ◽

False Negative ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

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Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

Scientific Reports ◽

10.1038/s41598-021-88625-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chukwunonso Onyilagha ◽

Henna Mistry ◽

Peter Marszal ◽

Mathieu Pinette ◽

Darwyn Kobasa ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Real Time ◽

Point Of Care ◽

Middle Eastern ◽

Turnaround Time ◽

Cross Reactivity ◽

Clinical Samples ◽

Diarrhea Virus ◽

Highly Sensitive ◽

Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

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Evaluation of Amplification Targets for the Specific Detection ofBordetella pertussisUsing Real-Time Polymerase Chain Reaction

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2014/763128 ◽

2014 ◽

Vol 25 (4) ◽

pp. 217-221 ◽

Cited By ~ 7

Author(s):

Mohammad Rubayet Hasan ◽

Rusung Tan ◽

Ghada N Al-Rawahi ◽

Eva Thomas ◽

Peter Tilley

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Target Genes ◽

Reference Panel ◽

Superior Performance ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Chain Reaction ◽

Pcr Assay ◽

Polymerase Chain

BACKGROUND:Bordetella pertussisinfections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detectB pertussisare typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.METHODS: A novelB pertussisreal-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481,ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including differentBordetellaspecies and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.RESULTS: Analytical sensitivity was highest for the assay targeting the IS481element; however, the assay lacked specificity forB pertussisin the reference panel and in the clinical samples. False-positive results were also observed with assays targeting theptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains ofB pertussis. However, a novel assay targeting the porin gene demonstrated high specificity forB pertussisboth in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted allB pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.CONCLUSION: A novel porin assay forB pertussisdemonstrated superior performance and may be useful for improved molecular detection ofB pertussisin clinical specimens.

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Comparison Between Broad-Range Real-Time and a Broad Range End-Point PCR Assays for the Detection of Bacterial 16S rDNA in Clinical Samples: A 1-Year Prospective Study in Routine Practice at the University Hospital of Strasbourg, France

American Journal of Clinical Pathology ◽

10.1093/ajcp/142.suppl1.163 ◽

2014 ◽

Vol 142 (suppl_1) ◽

pp. A163-A163 ◽

Cited By ~ 1

Author(s):

Mariam Meddeb ◽

Christelle Koebel ◽

Benoit Jaulhac ◽

Frédéric Schramm

Keyword(s):

Prospective Study ◽

Real Time ◽

16S Rdna ◽

University Hospital ◽

Clinical Samples ◽

Routine Practice ◽

End Point ◽

Pcr Assays ◽

The University

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Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

Journal of Medical Microbiology ◽

10.1099/jmm.0.010587-0 ◽

2009 ◽

Vol 58 (7) ◽

pp. 878-883 ◽

Cited By ~ 11

Author(s):

Wafa Habbal ◽

Fawza Monem ◽

Barbara C. Gärtner

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Isolates ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Pcr Product ◽

Highly Sensitive ◽

Primer Sets ◽

Analytical Sensitivities ◽

Strain Ad169

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993–2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

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