Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

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Rapid Detection of Equine Piroplasms Using Multiplex PCR and First Genetic Characterization of Theileria haneyi in Egypt

Pathogens ◽

10.3390/pathogens10111414 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1414

Author(s):

Bassma S. M. Elsawy ◽

Ahmed M. Nassar ◽

Heba F. Alzan ◽

Raksha V. Bhoora ◽

Sezayi Ozubek ◽

...

Keyword(s):

Multiplex Pcr ◽

Genetic Characterization ◽

Simultaneous Detection ◽

18S Rrna Gene ◽

Rrna Gene ◽

Theileria Equi ◽

Babesia Caballi ◽

Pcr Targeting ◽

First Time

Equine Piroplasmosis (EP) is an infectious disease caused by the hemoprotozoan parasites Theileria equi, Babesia caballi, and the recently identified species T. haneyi. Hereby, we used a multiplex PCR (mPCR) targeting the 18S rRNA gene of T. equi and B. caballi for the simultaneous detection of EP in Egyptian equids and examined the presence of T. haneyi infections in Egypt. Blood samples from 155 equids (79 horses and 76 donkeys) collected from different governorates of Egypt were examined by mPCR and PCR targeting T. hayeni. The mPCR method revealed a prevalence of T. equi of 20.3% in horses and of 13.1% in donkeys and a prevalence of B. caballi of 1.2% in horses. B. caballi was not detected in donkeys in the current study. The mPCR method also detected coinfections with both species (2.5% and 1.3% in horses and donkeys, respectively). Additionally, we report the presence of T. haneyi in Egypt for the first time in 53.1% of the horse and 38.1% of the donkey tested samples. Coinfection with T. haneyi and T. equi was found in 13.5% of the samples, while infection with the three EP species was found in 1.9% of the samples.

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Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

PLoS ONE ◽

10.1371/journal.pone.0255914 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0255914

Author(s):

Monia Ardhaoui ◽

Emna Ennaifer ◽

Anna Christina De Matos Salim ◽

Flávio Marcom Gomez ◽

Thalja Laasili ◽

...

Keyword(s):

Nested Pcr ◽

High Sensitivity ◽

Epidemiological Studies ◽

Multiple Infection ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Hpv Genotyping ◽

Genotyping Method ◽

Specificity And Sensitivity ◽

Hpv Types

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

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Molecular and microscopic detection of Babesia caballi and Theileria equi among working horses and donkeys in Cairo and Giza provinces of Egypt

10.21203/rs.3.rs-757240/v1 ◽

2021 ◽

Author(s):

Ahmed M. Soliman ◽

Nagwa M. Elhawary ◽

Nashwa M. Helmy ◽

Sahar M. Gadelhaq

Keyword(s):

Ixodid Tick ◽

Economic Losses ◽

Age Group ◽

Health Issues ◽

Conventional Pcr ◽

Theileria Equi ◽

Babesia Caballi ◽

Tick Borne Disease ◽

The Difference ◽

Age And Sex

Abstract Equine piroplasmosis (EP) is an ixodid tick-borne disease caused by Theileria equi and/or Babesia caballi that can lead to severe health issues and economic losses among equine population. This study aimed to determine the prevalence of T. equi and B. caballi among Egyptian equines based on microscopy and conventional PCR. Also, to determine the effect of season, age, and sex of on their prevalence and determining the difference in sensitivity between microscopy and conventional PCR in the diagnosis of EP. This study was carried out on 432 blood samples randomly collected from 146 horses and 286 donkeys during a period from April 2016 to March 2018. Microscopic examination revealed that among horses, 13 (8.9%) and 4 (2.7%) were infected by T. equi and B. caballi respectively. While among donkeys, 22 (7.7%), 16 (5.6%) respectively. While mixed infections were detected in 4 (1.4%) donkeys. There was a statistically nonsignificant relation between prevalence of infection and season and sex of equines but the highest prevalence was recorded in age group less than 5 years old. By conventional PCR, among 64 horses, 15 (23.4%) and 8 (12.5%) were infected by T. equi and B. caballi, respectively. While among 76 donkeys, 36 (47.4%), 16 (21.1%), and 5 (6.6%) were infected by T. equi, B. caballi, and mixed infection, respectively. Our finding proved the existence of T. equi and B. caballi among equines.

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Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovorain Asymptomatic Plant Material

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.2071-2078.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 2071-2078 ◽

Cited By ~ 84

Author(s):

Pablo Llop ◽

Anna Bonaterra ◽

Javier Peñalver ◽

María M. López

Keyword(s):

Nested Pcr ◽

Plant Material ◽

High Sensitivity ◽

Epidemiological Studies ◽

Sample Volume ◽

Small Sample ◽

Asymptomatic Plant ◽

Specificity And Sensitivity ◽

Closed Tube ◽

Pure Cultures

ABSTRACT A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

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Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

Journal of Clinical Microbiology ◽

10.1128/jcm.02883-13 ◽

2013 ◽

Vol 52 (2) ◽

pp. 557-563 ◽

Cited By ~ 29

Author(s):

X. Wang ◽

D. J. Seo ◽

M. H. Lee ◽

C. Choi ◽

A. B. Onderdonk

Keyword(s):

Multiplex Pcr ◽

Rapid Detection ◽

Isothermal Amplification ◽

Conventional Pcr ◽

Loop Mediated Isothermal Amplification ◽

Pcr Multiplex

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Evaluation of Production Lots of a Rapid Point-of-Care Lateral Flow Serological Test Intended for Identification of IgM and IgG against the N-Terminal Part of the Spike Protein (S1) of SARS-CoV-2

Viruses ◽

10.3390/v13061043 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1043

Author(s):

Tove Hoffman ◽

Linda Kolstad ◽

Bengt Rönnberg ◽

Åke Lundkvist

Keyword(s):

Predictive Value ◽

Serological Test ◽

Point Of Care ◽

External Validation ◽

High Sensitivity ◽

Lateral Flow ◽

Spike Protein ◽

Individual Level ◽

Lateral Flow Test ◽

Production Errors

The potential of rapid point-of-care (POC) tests has been subject of doubt due to an eventual risk of production errors. The aim was therefore to evaluate the two separate production lots of a commercial POC lateral flow test, intended for the detection of IgM and IgG against the SARS-CoV-2 spike protein (S1). Control samples consisted of serum from individuals with confirmed SARS-CoV-2 infection and pre-COVID-19 negative sera gathered from a biobank. The presence of anti-S1 IgM/IgG in the sera was verified by an in-house Luminex-based serological assay (COVID-19 SIA). One hundred samples were verified as positive for anti-S1 IgG and 74 for anti-S1 IgM. Two hundred samples were verified as negative for anti-S1 IgM/IgG. For the two lots of the POC-test, the sensitivities were 93.2% and 87.8% for IgM and 93.0% and 100% for IgG. The specificities were 100% for IgM and 99.5% for IgG. The positive predictive value was 100% for IgM and 98.9% and 99.0% for IgG. The negative predictive value was 97.6% and 95.7% for IgM, and 96.6% and 100% for IgG. The evaluated POC-test is suitable to assess anti-SARS-CoV-2 S1 IgM and IgG, as a measure of previous virus exposure on an individual level. The external validation of separate lots of rapid POC-tests is encouraged to ensure high sensitivity before market introduction.

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The associations between whole grain and refined grain intakes and serum C-reactive protein

European Journal of Clinical Nutrition ◽

10.1038/s41430-021-00996-1 ◽

2021 ◽

Author(s):

Riikka E. Taskinen ◽

Sari Hantunen ◽

Tomi-Pekka Tuomainen ◽

Jyrki K. Virtanen

Keyword(s):

Disease Risk ◽

High Sensitivity ◽

Epidemiological Studies ◽

Whole Grains ◽

Dietary Factors ◽

Low Grade ◽

Dietary Intakes ◽

Whole Grain ◽

Refined Grains ◽

Hs Crp

Abstract Background/objectives Epidemiological studies suggest that whole grain intake has inverse associations with low-grade inflammation, but findings regarding refined grains are inconclusive. Our objective was to investigate whether consumption of whole or refined grains is associated with serum high sensitivity CRP (hs-CRP). Subjects/methods The study included 756 generally healthy men and women aged 53–73 years from the Kuopio Ischaemic Heart Disease Risk Factory Study, examined in 1999–2001. Dietary intakes were assessed using 4-day food records. ANCOVA and linear regression were used for analyses. Results The mean intake of whole and refined grains was 136 g/day (SD 80) and 84 g/day (SD 46), respectively. Higher whole grain intake was associated with lower hs-CRP concentration and higher refined grain intake with higher concentration after adjustment for lifestyle and dietary factors. Each 50 g/d higher whole grain intake was associated with 0.12 mg/L (95% Cl 0.02–0.21 mg/L) lower hs-CRP concentration and each 50 g/d higher refined grain intake with 0.23 mg/L (95% Cl 0.08–0.38) higher concentration. Adjustment for fibre from grains attenuated the associations especially with whole grains. There were no statistically significant interactions according to gender or BMI (P for interactions >0.065). Conclusions The results of this study suggest that higher intake of whole grains is associated with lower concentrations of hs-CRP and higher intake of refined grains is associated with higher concentrations. However, especially the association with whole grain intake was attenuated after adjusting for fibre intake from grains, suggesting that cereal fibre may partly explain the association.

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