scholarly journals A Comparison of PCR and ELISA Methods to Detect Different Stages of Plasmodium Vivax in Anopheles Arabiensis.

2021 ◽  
Author(s):  
Allison L Hendershot ◽  
Endashaw Esayas ◽  
Alice C. Sutcliffe ◽  
Seth R. Irish ◽  
Endalamaw Gadisa ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Post Infection ◽  
Anopheles Arabiensis ◽  
Life Stage ◽  
Malaria Epidemiology ◽  
Highly Sensitive ◽  
Pcr Method ◽  
Infection Stage ◽  
I Gene ◽  
Time Point

Abstract Background: In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods: A PCR-based method targeting the Plasmodium COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection timepoints (days 0.5, 1, 6, 9, 12, 15). Head and thoraces, and abdomens for each specimen were tested separately with both methods. Agreement between methods at each infection stage was measured using Cohen’s Kappa measure of test association.Results: Infection status of mosquitoes was assessed in approximately 90 head and thoraces and 90 abdomens at each time point; in total 538 head and thoraces and 534 abdomens were tested. In mosquitoes bisected after 0.5, 1-, and 6-days post-infection, the COX-I PCR detected Plasmodium DNA in both the abdomens (88%, 78%, and 67%, respectively) and head and thoraces (69%, 60%, and 44%, respectively) whilst CSP-ELISA detect sporozoites in only 1 abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9-, 12-, and 15-days post-infection in both the head and thoraces and abdomens. There was fair agreement between both methods for time points 9 -15 days post-infection (κ = 0.312, 95% CI: 0.230 – 0.394). Conclusion: The COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the COX-I PCR is a poor candidate for identifying infectious mosquitoes.

scholarly journals A comparison of PCR and ELISA methods to detect different stages of Plasmodium vivax in Anopheles arabiensis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Allison L. Hendershot ◽  
Endashaw Esayas ◽  
Alice C. Sutcliffe ◽  
Seth R. Irish ◽  
Endalamaw Gadisa ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Post Infection ◽  
Anopheles Arabiensis ◽  
Life Stage ◽  
Time Points ◽  
Malaria Epidemiology ◽  
Highly Sensitive ◽  
Pcr Method ◽  
Infection Stage ◽  
Method Agreement

Abstract Background In characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s mitochondrial (mt) cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito. Methods A PCR-based method targeting the Plasmodium mt COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection time points (days 0.5, 1, 6, 9, 12, 15). The head and thorax and the abdomen for each specimen were tested separately with each method. Agreement between methods at each infection stage was measured using Cohen’s kappa measure of test association. Results Infection status of mosquitoes was assessed in approximately 90 head/thorax and 90 abdomen segments at each time point; in total, 538 head/thorax and 534 abdomen segments were tested. In mosquitoes bisected after 0.5, 1, and 6 days post-infection (dpi), the mt COX-I PCR detected Plasmodium DNA in both the abdomen (88, 78, and 67%, respectively) and head/thorax segments (69, 60, and 44%, respectively), whilst CSP ELISA detected sporozoites in only one abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9, 12, and 15 dpi in both the head and thorax and abdomen. There was fair agreement between methods for time points 9–15 dpi (κ = 0.312, 95% CI: 0.230–0.394). Conclusions The mt COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the mt COX-I PCR is a poor candidate for identifying infectious mosquitoes. Graphical Abstract

DNA Barcoding and Phylogenetic Analysis Sugarcane (Saccharum officinarum L.) Based on matK ( maturase K) Gene

2020 ◽  
Vol 840 ◽  
pp. 162-170
Author(s):  
Ganies Riza Aristya ◽  
Fauzana Putri ◽  
Rina Sri Kasiamdari ◽  
Arni Musthofa
Keyword(s):  
Phylogenetic Tree ◽  
Molecular Level ◽  
Economic Value ◽  
Saccharum Officinarum ◽  
Saccharum Spontaneum ◽  
Dna Barcodes ◽  
Pcr Method ◽  
Hybrid Cultivar ◽  
I Gene ◽  
Sequencing Process

Sugarcane (Saccharum officinarum L.) is an agricultural commodities with a great extent of diversity and high economic value. In Indonesia, the great extent of diversity of sugarcane is evidenced by a large number of cultivars cultivated. Sugarcane diversities at the molecular level can be seen using DNA barcodes, one of which is the matK. The purpose of the study was to identify and characterize matK and reconstruct the phylogenetic tree to determine the phylogeny of 24 sugarcane cultivars Indonesia. matK was amplified using the PCR method with matK F-5’ATGATTAATTAAGAGTAAGAGGAT-3’ and matK R-5’AATGCAAAAATTCGAAGGGT-3. Results showed that the matK gene was successfully amplified as many as 1531 bp. The sequencing process was done to determine the nucleotide sequence and compared with those of the GenBank database. It showed that the samples used had a similarity of 98.87%-99.44% to that of matK in Saccharum officinarum, Saccharum hybrid cultivar and Saccharum spontaneum. Reconstruction of the phylogenetic tree showed that the samples used were located in the same clade with a zero genetic distance, while all the references from NCBI were also located in the same clade. The analysis of genetic variation indicated that it had no haplotype value.

scholarly journals RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course

2021 ◽  
Vol 8 ◽  
Author(s):  
Kirsten E. McLoughlin ◽  
Carolina N. Correia ◽  
John A. Browne ◽  
David A. Magee ◽  
Nicolas C. Nalpas ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Mycobacterium Bovis ◽  
Peripheral Blood ◽  
Post Infection ◽  
Time Course ◽  
De Novo ◽  
Differentially Expressed Gene ◽  
Differentially Expressed ◽  
Infection Time ◽  
Time Point

Bovine tuberculosis, caused by infection with members of the Mycobacterium tuberculosis complex, particularly Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including RNA sequencing, has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analysed the transcriptome of bovine whole peripheral blood samples collected at −1 week pre-infection and +1, +2, +6, +10, and +12 weeks post-infection time points. Differentially expressed genes were catalogued and evaluated at each post-infection time point relative to the −1 week pre-infection time point and used for the identification of putative candidate host transcriptional biomarkers for M. bovis infection. Differentially expressed gene sets were also used for examination of cellular pathways associated with the host response to M. bovis infection, construction of de novo gene interaction networks enriched for host differentially expressed genes, and time-series analyses to identify functionally important groups of genes displaying similar patterns of expression across the infection time course. A notable outcome of these analyses was identification of a 19-gene transcriptional biosignature of infection consisting of genes increased in expression across the time course from +1 week to +12 weeks post-infection.

scholarly journals Detectability of Pseudomonassyringae pv. aesculi from European Horse Chestnut Using Quantitative PCR Compared with Traditional Isolation

Forests ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1062
Author(s):  
Salome Schneider ◽  
Christopher Schefer ◽  
Joana Beatrice Meyer
Keyword(s):  
Quantitative Pcr ◽  
Bacterial Growth ◽  
Target Gene ◽  
Horse Chestnut ◽  
Inoculation Experiment ◽  
Copy Numbers ◽  
Infection Stage ◽  
Time Point

Bleeding cankers on horse chestnut trees (Aesculushippocastanum and Aesculus × carnea), caused by Pseudomonassyringae pv. aesculi, have been reported across Europe. In the present study, we show the successful detection of P. syringae pv. aesculi on symptomatic horse chestnut trees in Switzerland using quantitative PCR (qPCR). However, P. syringae pv. aesculi was also detected by qPCR on trees from which no isolate was obtained through cultivation. Reduced isolation success and low copy numbers of the target gene were correlated with the increasing age of symptomatic horse chestnut trees. The potential of detecting non-viable P. syringae pv. aesculi by qPCR was evaluated using an inoculation experiment with dead bacteria and detection by qPCR and cultivation. The detectability of DNA from P. syringae pv. aesculi cells dropped by 34.5% one day after inoculation and then decreased only slightly until the end of the experiment (22 days after inoculation). In contrast, no bacterial growth was observed at any time point after the inactivation of the bacteria. To protect horse chestnut trees, evaluating the viability and actual infection stage of the bacterium may play an important role.

scholarly journals Evaluation of Putative Type II Toxin-Antitoxin Systems and Lon Protease Expression in Shigella flexneri Following Infection of Caco-2 Cells

2020 ◽  
Vol 15 (3) ◽  
Author(s):  
Erfan Kheradmand ◽  
Shabnam Razavi ◽  
Malihe Talebi ◽  
Mahmood Jamshidian
Keyword(s):  
Cell Line ◽  
Post Infection ◽  
Shigella Flexneri ◽  
Fold Increase ◽  
Expression Level ◽  
Bacterial Invasion ◽  
P Value ◽  
Lon Protease ◽  
Expression Levels ◽  
Pcr Method

: Shigella flexneri causes bacillary dysentery in developing countries. Due to recent reports regarding antimicrobial resistance in human S. flexneri, finding alternative therapeutics is of vital importance. Toxin-antitoxin (TA) systems have recently been introduced as antimicrobial targets owing to their involvement in bacterial survival in stress conditions and “persister” cell formation. In this study, the presence of four TA loci were studied in S. flexneri ATCC 12022. The presence of genes coding for the identified TA loci and Lon protease were confirmed by the PCR method using specific primers. Caco-2 cell lines were then infected with this standard strain, and 8 and 24 h post-infection, expression levels of genes coding for the studied TA loci, and Lon protease were evaluated using a real-time PCR method. Expression of mazF, GNAT (Gcn5-related N-acetyltransferase), yeeU, pfam13975, and Lon genes showed 5.4, 9.8, 2.3, 2.7, and 13.8-fold increase, respectively, 8 h after bacterial invasion of the Caco-2 cell line. In addition, the expression of the aforementioned genes showed 4.8, 10.8, 2.3, 3.7, and 16.8-fold increase after 24 h. The GNAT and lon genes showed significantly higher expression levels compared to the control (P value < 0.05). However, the increase in the expression level of yeeU was the same at 8 h and 24 h post-infection. In addition, mazF expression level showed a slight decrease at 24 h compared to 8h post-infection. Genes coding for GNAT and Lon protease showed a significantly higher expression after invading the Caco-2 cell line. Therefore, targeting GNAT or Lon protease can be taken into consideration for finding novel antimicrobial drug strategies. The exact functions and mechanisms of TA systems in S. flexneri isolates are suggested to be experimentally determined.

Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR

2004 ◽  
Vol 53 (12) ◽  
pp. 1207-1214 ◽  
Author(s):  
Ralf Gutzmer ◽  
Susanne Mommert ◽  
Uta Küttler ◽  
Thomas Werfel ◽  
Alexander Kapp
Keyword(s):  
Skin Diseases ◽  
Rapid Identification ◽  
Diagnostic Information ◽  
Clinical Samples ◽  
Melting Points ◽  
Number Of Patients ◽  
Highly Sensitive ◽  
Fungal Dna ◽  
Pcr Method ◽  
Primer Sets

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

Expression Profile of Prophenoloxidase-Encoding (acppo6) Gene of Plasmodium vivax-Refractory Strain of Anopheles culicifacies

2010 ◽  
Vol 47 (6) ◽  
pp. 1220-1226 ◽  
Author(s):  
Anil Sharma ◽  
Janneth Rodrigues ◽  
Mayur K. Kajla ◽  
Neema Agrawal ◽  
T. Adak ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Expression Profile ◽  
Anopheles Culicifacies ◽  
I Gene

Effects of snail size on encystment of Echinostoma caproni in juvenile Biomphalaria glabrata (NMRI strain) and observations on the survival of infected snails

2004 ◽  
Vol 78 (3) ◽  
pp. 277-279 ◽  
Author(s):  
J.L. Schneck ◽  
B. Fried
Keyword(s):  
Post Infection ◽  
Biomphalaria Glabrata ◽  
Echinostoma Caproni ◽  
Individual Exposure ◽  
Shell Diameter ◽  
Time Point

AbstractThe effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7–1.1 mm shell diameter) and juvenile (2–3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.

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