scholarly journals Development of loop-mediated isothermal amplification (LAMP) assay for detection of Pseudocercospora angolensis in sweet orange

2021 ◽  
Author(s):  
Patricia Driciru ◽  
M Claire Mugasa ◽  
Robert Acidri ◽  
John Adriko
Keyword(s):  
Yield Loss ◽  
Sweet Orange ◽  
Lamp Assay ◽  
Effective Control ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Predictive Values ◽  
Fruit Spot ◽  
Orange Leaf ◽  
Polymerase Chain

AbstractPseudocercospora angolensis is the causative agent of Pseudocercospora leaf and fruit spot disease in citrus which can result in up to 100% yield loss. Early diagnosis of this disease is vital for effective control. This study aimed at developing a loop-mediated amplification (LAMP) system for detecting P. angolensis in sweet oranges in comparison with Polymerase Chain Reaction (PCR) and using microscopy as a gold standard. Twelve non-target species were used to assess the analytical specificity of LAMP and PCR whereas the analytical sensitivity was determined using serial dilutions of P. angolensis DNA. The diagnostic accuracies of the two assays were evaluated using DNA from 150 diseased and 50 non-diseased sweet orange leaf samples. The analytical sensitivity and detection time of LAMP were of 10−4 ng/ μl and 40 minutes, respectively. The analytical sensitivity of PCR was 10ng/μl and it was specific to P. angolensis whereas three relatives of P. angolensis were detectable by LAMP. The diagnostic sensitivities of LAMP (93%) and microscopy (100%) were significantly different (X2 = 8.38, P = 0.0038) unlike the diagnostic specificities (90%) and (100%), respectively (X2 = 3.37, P = 0.066). Microscopy was significantly more sensitive than PCR (32.6%) (X2 = 149.26, P < 2.2e-16) and equally specific as PCR (P=NA). The positive predictive values of PCR and LAMP were 100% and 96.5% respectively whereas the negative predictive values were 33.1% and 81.8% respectively. The LAMP assay developed in this study offers a great tool for routine screening sweet orange samples for P. angolensis.

scholarly journals Field Evaluation of the Performance of Two Rapid Diagnostic Tests for Meningitis in Niger and Burkina Faso

2021 ◽  
Vol 9 (4) ◽  
pp. 832 ◽  
Author(s):  
Marc Rondy ◽  
Mamadou Tamboura ◽  
Fati Sidikou ◽  
Issaka Yameogo ◽  
Kambire Dinanibe ◽  
...  
Keyword(s):  
Burkina Faso ◽  
Field Evaluation ◽  
Health Centers ◽  
Outbreak Detection ◽  
Health Staff ◽  
Chain Reaction ◽  
Predictive Values ◽  
Independent Evaluation ◽  
Polymerase Chain ◽  
Lateral Flow Tests

New lateral flow tests for the diagnosis of Neisseria meningitidis (Nm) (serogroups A, C, W, X, and Y), MeningoSpeed, and Streptococcus pneumoniae (Sp), PneumoSpeed, developed to support rapid outbreak detection in Africa, have shown good performance under laboratory conditions. We conducted an independent evaluation of both tests under field conditions in Burkina Faso and Niger, in 2018–2019. The tests were performed in the cerebrospinal fluid of suspected meningitis cases from health centers in alert districts and compared to reverse transcription polymerase chain reaction tests performed at national reference laboratories (NRLs). Health staff were interviewed about feasibility. A total of 327 cases were tested at the NRLs, with 26% confirmed Nm (NmC 63% and NmX 37%) and 8% Sp. Sensitivity and specificity were, respectively, 95% (95% CI: 89–99) and 90% (95% CI: 86–94) for Nm and 92% (95% CI: 75–99) and 99% (95% CI: 97–100) for Sp. Positive and negative predictive values were, respectively, 77% (95% CI: 68–85) and 98% (95% CI: 95–100) for Nm and 86% (95% CI: 67–96) and 99% (95% CI: 98–100) for Sp. Concordance showed 82% agreement for Nm and 97% for Sp. Interviewed staff evaluated the tests as easy to use and to interpret and were confident in their readings. Results suggest overall good performance of both tests and potential usefulness in meningitis outbreak detection.

scholarly journals Occurrence and Identification of a New Vector of Rice Orange Leaf Phytoplasma in South China

Plant Disease ◽  
2015 ◽  
Vol 99 (11) ◽  
pp. 1483-1487 ◽  
Author(s):  
Shu Li ◽  
Weijia Hao ◽  
Guanghua Lu ◽  
Jilei Huang ◽  
Chuanhe Liu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
South China ◽  
Rice Tungro Bacilliform Virus ◽  
Crop Failure ◽  
Chain Reaction ◽  
Phloem Tissue ◽  
Poor Growth ◽  
Different Seasons ◽  
Orange Leaf ◽  
Polymerase Chain

Rice orange leaf disease (ROLD) is caused by rice orange leaf phytoplasma (ROLP) and occurs sporadically in rice-growing areas in countries of eastern and southeastern Asia. ROLD caused severe damage to rice production in South China in the 1980s. Although its impact subsequently declined in South China, it has reemerged as a serious threat recently. Our study showed that ROLD occurrence varies in different seasons and fields. It was more severe in summer-grown crops (from July to October) than in spring-grown crops (from March to July). In most fields, the incidence was less than 10%, and diseased plants were scattered throughout the fields. In 20% of fields, the incidence was between 10 and 30%. In some fields, over 90% of plants were affected, causing crop failure. Typical symptoms of ROLD include orange-colored leaves and poor growth. Diseased plants were determined as positive for ROLP but negative for Rice tungro bacilliform virus, Rice tungro spherical virus, and Rice transitory yellowing virus through polymerase chain reaction and reverse-transcription polymerase chain reaction. Phytoplasma bodies but not virus-like particles were observed by electron microscopy in phloem tissue of diseased leaves. The leafhopper Inazuma dorsalis, previously identified as the unique vector for ROLP, was rare in the affected fields. Another leafhopper, Nephotettix cincticeps, previously considered a nonvector for this phytoplasma, was very common. Transmission tests revealed that this insect could also transmit ROLP; therefore, it might represent a new vector responsible for the recent incidence of ROLD.

scholarly journals Polymerase Chain Reaction (PCR) as a Potential Point of Care Laboratory Test for Leprosy Diagnosis—A Systematic Review

2018 ◽  
Vol 3 (4) ◽  
pp. 107 ◽  
Author(s):  
Sushma Tatipally ◽  
Aparna Srikantam ◽  
Sanjay Kasetty
Keyword(s):  
Systematic Review ◽  
Polymerase Chain Reaction ◽  
Point Of Care ◽  
Effective Control ◽  
Chain Reaction ◽  
Review Analysis ◽  
Pubmed Database ◽  
Early Case ◽  
Polymerase Chain ◽  
Clinical Conditions

Leprosy is an infectious disease caused by Mycobacterium leprae and mainly affects skin, peripheral nerves, and eyes. Suitable tools for providing bacteriological evidence of leprosy are needed for early case detection and appropriate therapeutic management. Ideally these tools are applicable at all health care levels for the effective control of leprosy. This paper presents a systematic review analysis in order to investigate the performance of polymerase chain reaction (PCR) vis-à-vis slit skin smears (SSS) in various clinical settings and its potential usefulness as a routine lab test for leprosy diagnosis. Records of published journal articles were identified through PubMed database search. Twenty-seven articles were included for the analysis. The evidence from this review analysis suggests that PCR on skin biopsy is the ideal diagnostic test. Nevertheless, PCR on SSS samples also seems to be useful with its practical value for application, even at primary care levels. The review findings also indicated the necessity for improving the sensitivity of PCR and further research on specificity in ruling out other clinical conditions that may mimic leprosy. The M. leprae-specific repetitive element (RLEP) was the most frequently-used marker although its variable performance across the clinical sites and samples are a matter of concern. Undertaking further research studies with large sample numbers and uniform protocols studied simultaneously across multiple clinical sites is recommended to address these issues.

scholarly journals Cluster of Severe Acute Respiratory Syndrome Coronavirus 2 Infections Linked to Music Clubs in Osaka, Japan

2020 ◽  
Vol 222 (10) ◽  
pp. 1635-1640
Author(s):  
Nobuhiko Sugano ◽  
Wataru Ando ◽  
Wakaba Fukushima
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Personal Hygiene ◽  
Effective Control ◽  
Chain Reaction ◽  
Secondary Transmission ◽  
Live Music ◽  
Polymerase Chain ◽  
Mode Of Transmission ◽  
Index Cases ◽  
Epidemiological Information

Abstract Background It is important to understand the mode of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for disease control. We aimed to clarify how soon SARS-CoV-2 transmission can occur after infection by asymptomatically infected individuals. Methods We analyzed the publicly available epidemiological information for a cluster of 108 cases of coronavirus disease 2019 (COVID-19) cases in Osaka, Japan. Results Among cases, 51 cases attended a live music club only once and were considered to have a single visit. Ten remained asymptomatic at the time of COVID-19 diagnosis by reverse-transcription polymerase chain reaction, which was on average 20 days after exposure. Three routes of secondary transmission were identified, with 2–4 days from infection to transmission. All index cases for secondary transmission were asymptomatic at the time of contact with other people. Based on the date of symptom onset in the remaining 41 cases, the period from exposure to illness ranged from 2 to 17 days. Conclusions Seemingly healthy people could spread SARS-CoV-2 during intense activities in enclosed environments without sufficient ventilation. Asymptomatically infected persons can transmit the virus as soon as 2 days after infection. Continuous efforts to avoid crowding and to maintain personal hygiene are needed for effective control of COVID-19.

scholarly journals Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

2014 ◽  
Vol 26 (6) ◽  
pp. 755-760 ◽  
Author(s):  
Maria J. Clavijo ◽  
Simone Oliveira ◽  
Jeffrey Zimmerman ◽  
Aaron Rendahl ◽  
Albert Rovira
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Systemic Disease ◽  
Analytical Sensitivity ◽  
Upper Respiratory Tract ◽  
Chain Reaction ◽  
Mycoplasma Hyorhinis ◽  
Field Samples ◽  
Polymerase Chain

Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

scholarly journals Performance of a Commercial Polymerase Chain Reaction Test for EndocervicalChlamydia trachomatisInfection in a University Hospital Population

1998 ◽  
Vol 6 (5) ◽  
pp. 224-229
Author(s):  
C. H. Livengood III ◽  
K. A. Boggess ◽  
J. W. Wrenn ◽  
A. P. Murtha
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Test ◽  
Laboratory Evaluation ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Predictive Values ◽  
Target Dna ◽  
Wide Range ◽  
Polymerase Chain ◽  
Pcr Test

Objectives:To examine the accuracy of a commercial polymerase chain reaction (PCR) test (Amplicor CTR, Roche Diagnostic Systems, Branchburg NJ) for identification of endocervical chlamydial infections through both laboratory evaluation and among a diverse teaching hospital patient population.Methods:Testing of reliable threshold inocula and reproducibility were carried out using laboratory stock organisms. Paired endocervical samples from patients with a wide range of indications were tested by PCR and an established culture procedure, and discrepant pairs were further analyzed to determine true results.Results:Laboratory evaluation suggested that one copy of target DNA from a viable organism consistently yielded a positive result, and test reproducibility was very good, with an overall coefficient of variation of 15%. Compared to true results in 1,588 paired clinical samples from 1,489 women with a 10% prevalence of infection, the PCR test and culture yielded respective sensitivities of 87.4% and 78.0%, and negative predictive values of 98.6% and 97.6%. Specificity and positive predictive value for both tests were 100%. Cost per specimen was nearly identical at $18.84 and $18.88 respectively. Polymerase inhibitors and organisms lacking target DNA were not found in false-negative PCR samples.Conclusion:This commercial PCR test is accurate, cost-competitive, and much faster than culture for diagnosis of endocervical chlamydia infections in our population of intermediate prevalence of chlamydial infection.

Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction

2020 ◽  
Vol 58 (4) ◽  
pp. 527-532 ◽  
Author(s):  
Jee-Soo Lee ◽  
Miyoung Kim ◽  
Moon-Woo Seong ◽  
Han-Sung Kim ◽  
Young Kyung Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Circulating Tumor Dna ◽  
Analytical Sensitivity ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Specimen Type ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Ctdna Analysis ◽  
Tumor Dna

AbstractBackgroundChoosing the specimen type is the first step of the pre-analytical process. Previous reports suggested plasma as the optimal specimen for circulating tumor DNA (ctDNA) analysis. However, head-to-head comparisons between plasma and serum using platforms with high analytical sensitivity, such as droplet digital polymerase chain reaction (ddPCR), are limited, and several recent studies have supported the clinical utility of serum-derived ctDNA. This study aimed to compare the DNA profiles isolated from plasma and serum, characterize the effects of the differences between specimens on ctDNA measurement, and determine the major contributors to these differences.MethodsWe isolated cell-free DNA (cfDNA) from 119 matched plasma/serum samples from cancer patients and analyzed the cfDNA profiles by DNA fragment sizing. We then assessed KRAS mutations in ctDNA from matched plasma/serum using ddPCR.ResultsThe amount of large DNA fragments was increased in serum, whereas that of cfDNA fragments (<800 bp) was similar in both specimens. ctDNA was less frequently detected in serum, and the KRAS-mutated fraction in serum was significantly lower than that in plasma. The differences in ctDNA fractions between the two specimen types correlated well with the amount of large DNA fragments and white blood cell and neutrophil counts.ConclusionsOur results provided detailed insights into the differences between plasma and serum using DNA fragment sizing and ddPCR, potentially contributing to ctDNA analysis standardization. Our study also suggested that using plasma minimizes the dilution of tumor-derived DNA and optimizes the sensitivity of ctDNA analysis. So, plasma should be the preferred specimen type.

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

2014 ◽  
Vol 25 (4) ◽  
pp. 217-221 ◽  
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.

Polymerase chain reaction for the amplification of the 121-bp repetitive sequence ofSchistosoma mansoni: a highly sensitive potential diagnostic tool for areas of low endemicity

2014 ◽  
Vol 89 (6) ◽  
pp. 769-773 ◽  
Author(s):  
E. Ferrer ◽  
F. Pérez ◽  
I. Bello ◽  
A. Bolívar ◽  
M. Lares ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Repetitive Sequence ◽  
Annealing Temperature ◽  
Immunological Methods ◽  
Molecular Technique ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Highly Sensitive ◽  
Polymerase Chain

AbstractSchistosomiasis is a disease caused by parasitic flatworms of the genusSchistosoma, whose diagnosis has limitations, such as the low sensitivity and specificity of parasitological and immunological methods, respectively. In the present study an alternative molecular technique requiring previous standardization was carried out using the polymerase chain reaction (PCR) for the amplification of a 121-bp highly repetitive sequence forSchistosoma mansoni.DNA was extracted from eggs ofS. mansoniby salting out. Different conditions were standardized for the PCR technique, including the concentration of reagents and the DNA template, annealing temperature and number of cycles, followed by the determination of the analytical sensitivity and specificity of the technique. Furthermore, the standardized PCR technique was employed in DNA extracted, using Chelex®100, from samples of sera of patients with an immunodiagnosis of schistosomiasis. The optimal conditions for the PCR were 2.5 mmMgCl2, 150 mmdeoxynucleoside triphosphates (dNTPs), 0.4 μmprimers, 0.75 U DNA polymerase, using 35 cycles and an annealing temperature of 63°C. The analytical sensitivity of the PCR was 10 attograms of DNA and the specificity was 100%. The DNA sequence was successfully detected in the sera of two patients, demonstrating schistosomiasis transmission, although low, in the community studied. The standardized PCR technique, using smaller amounts of reagents than in the original protocol, is highly sensitive and specific for the detection of DNA fromS. mansoniand could be an important tool for diagnosis in areas of low endemicity.

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