scholarly journals PCR Detection of Mixed and Zoonoses Malaria Using Plasmodium spp Dynein Light Chain (dlc-tctex) Gene

2019 ◽  
pp. 1-12
Author(s):  
Maureen W. Kariuki ◽  
Elijah K. Githui ◽  
Andrew G. McArthur ◽  
Rashid A. Aman ◽  
Nyamu M. Njagi ◽  
...  
Keyword(s):  
Malaria Diagnosis ◽  
Clinical Malaria ◽  
Cross Reactivity ◽  
Pcr Detection ◽  
Clinical Samples ◽  
Dynein Light Chain ◽  
Pcr Assay ◽  
Specific Primers ◽  
Dynein Light Chains ◽  
Species Specific

Novel gene targets are needed in accurate diagnosis of malaria. Previous studies show that the dynein light chains (dlc) in Plasmodium are uniquely conserved within the species, possibly due to their role as the cargo adptor moiety. This study aimed at the development of PCR assay for the detection of Plasmodium based on the (dlc-Tctex) as a genus and species-specific tool in malaria diagnosis. Multiple primers were designed based on Plasmodium spp dlc(Tctex) genes. The primers were applied on PCR to detect malaria on clinical samples and on laboratory maintained isolates of P. falciparum and P. vivax for human infecting species and P. knowlesi and P. cynomolgi for zoonoses infection involving primates. The amplified PCR fragments were gene cleaned and sequenced. BLASTn e-values output from the raw nucleotide queries supports that the genes are uniquely conserved.  Species-specific primers amplified P.  falciparum infections with no cross-reactivity to P. vivax, P. knowlesi or P. cynomolgi species. In this assay only 11 out of the 30 microscope positive malaria positive clinical blood samples were positive for PCR detection of P. falciparum infection. Primers designed for Plasmodium genus amplified the target band in all clinical malaria samples but also had another specific band amplification. This preliminary data demonstrate that a species-specific dlc(Tctex) PCR assay can be used for detection of P. falciparum and optimized genus primers can be applied to differentiate mixed malaria infections.

scholarly journals Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 308
Author(s):  
Joana Carvalho-Pereira ◽  
Filipa Fernandes ◽  
Ricardo Araújo ◽  
Jan Springer ◽  
Juergen Loeffler ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Correct Identification ◽  
Colony Pcr ◽  
Pcr Multiplex ◽  
Pathogen Dna ◽  
Species Specific

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

scholarly journals Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

2004 ◽  
Vol 70 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Kado ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Intestinal Flora ◽  
Healthy Adults ◽  
Pcr Detection ◽  
Distribution Analysis ◽  
Specific Primers ◽  
Cell Counts ◽  
Species Specific

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Development of multiplex PCR for rapid detection of metallo-β-lactamase genes in clinical isolates of Acinetobacter baumannii

2020 ◽  
Author(s):  
Reza Ranjbar ◽  
Shahin Zayeri ◽  
Amir Mirzaie
Keyword(s):  
Acinetobacter Baumannii ◽  
Multiplex Pcr ◽  
Rapid Detection ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

Background and Objectives: Acinetobacter baumannii has been known as a major pathogen causing nosocomial infec- tions. The aim of this study was to develop multiplex PCR for rapid and simultaneous detection of metallo-β-lactamase (MBL) genes in clinical isolates of A. baumannii. Materials and Methods: In this study, we used three sets of primers to amplify the MBL genes including bla        ,     bla   and bla   OXA-48 . The multiplex PCR assay was optimized for rapid and simultaneous detection of MBL genes in A. bau-   OXA-23   NDM   mannii strains recovered from clinical samples. Results: A. baumannii strains recovered from clinical samples were subjected to the study. The multiplex PCR produced 3   OXA-48   OXA-23   bands of 501 bp for bla        , 744 bp for bla observed in multiplex PCR.   OXA-48   and 623 bp for bla   NDM   genes. In addition to, no any cross-reactivity was   Conclusion: Based on obtained data, the multiplex PCR had a good specificity without any cross reactivity and it appears that the multiplex PCR is reliable assay for simultaneous detection of MBL genes in A. baumannii strains.  

Specific PCR Detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in Chicken Meat

2009 ◽  
Vol 72 (7) ◽  
pp. 1491-1495 ◽  
Author(s):  
DANIELA PENTIMALLI ◽  
NICOLETTE PEGELS ◽  
TERESA GARCÍA ◽  
ROSARIO MARTÍN ◽  
ISABEL GONZÁLEZ
Keyword(s):  
Pcr Amplification ◽  
Chicken Meat ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Gyra Gene ◽  
Specific Primers ◽  
Specific Pcr ◽  
Arcobacter Butzleri ◽  
Species Specific

An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri–specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.

scholarly journals An alternative PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene

2021 ◽  
Author(s):  
Xi He ◽  
Derong Zhou ◽  
Yanwu Sun ◽  
Yuan Zhang ◽  
Xiaogang Zhang ◽  
...  
Keyword(s):  
Detection Method ◽  
Southern China ◽  
Acute Infection ◽  
High Sensitivity ◽  
High Specificity ◽  
Pcr Detection ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Pcr Method

Abstract Background Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. Methods To established a high specificity and sensitivity PCR detection method for swine toxoplasmosis, we used T. gondii GRA14 gene as target to design specific primers and established a PCR detection method for swine toxoplasmosis. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016–2017 were assessed by the newly established GRA14 gene PCR method. Result Altogether, we used T. gondii GRA14 gene as target to design specific primers and established a high specificity and sensitivity PCR detection method for swine toxoplasmosis; in particular, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii, and it could be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. The overall T. gondii infection rate was 18.9% (1033/5462) by the newly established GRA14 gene PCR method. According to statistical analysis among different regions, the positive rates of swine toxoplasmosis in the Shaanxi, Fujian and Guangdong areas in China from 2016 to 2017 were the highest, at 31.7% (44/139), 21.9% (86/391) and 18.8% (874/4645), respectively (χ2 = 84.2, P < 0.0001). Specimens collected in 2017 had a higher positive rate (19.1% or 886/4639) than those collected in 2016 (16.1% or 155/963) (χ2 = 4.5, P < 0.05). Specimens collected in autumn (39.4% or 187/474), spring (22.8% or 670/2940) and winter (18.2% or 129/709) also had higher positive rates than those collected in summer (3.8% or 57/1479) (χ2 = 427.7, P < 0.0001). Conclusions These results indicate that the new PCR method based on the T. gondii GRA14 gene would be useful for the diagnosis of swine toxoplasmosis and that it would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

scholarly journals Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains

1999 ◽  
Vol 37 (12) ◽  
pp. 3906-3911 ◽  
Author(s):  
Eugene J. Leys ◽  
James H. Smith ◽  
Sharon R. Lyons ◽  
Ann L. Griffen
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Sequences ◽  
Allelic Variation ◽  
Intergenic Spacer ◽  
Heteroduplex Analysis ◽  
Clinical Samples ◽  
Specific Primers ◽  
Intergenic Spacer Region ◽  
Species Specific ◽  
Multiple Strains

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types ofP. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivaliswas detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains ofP. gingivalis in large numbers of samples.

scholarly journals Application of pbp1A PCR in Identification of Penicillin-Resistant Streptococcus pneumoniae

1999 ◽  
Vol 37 (3) ◽  
pp. 628-632 ◽  
Author(s):  
Mignon du Plessis ◽  
Anthony M. Smith ◽  
Keith P. Klugman
Keyword(s):  
Streptococcus Pneumoniae ◽  
Clinical Isolates ◽  
Penicillin Resistance ◽  
Binding Region ◽  
Pcr Assay ◽  
Specific Primers ◽  
Predictive Values ◽  
Species Specific ◽  
Seminested Pcr

A seminested PCR assay, based on the amplification of the pneumococcal pbp1A gene, was developed for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 μg/ml) and higher-level (MICs = ≥1 μg/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of thepbp1A penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the pbp1A gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of ≥0.25 and ≥1 μg/ml, respectively. A total of 183 clinical isolates were tested with the pbp1A assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were ≥0.25 μg/ml and were both 100% for strains for which the MICs were ≥1 μg/ml.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

scholarly journals Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

2007 ◽  
Vol 97 (10) ◽  
pp. 1290-1297 ◽  
Author(s):  
Jennifer S. Falacy ◽  
Gary G. Grove ◽  
Walter F. Mahaffee ◽  
Heather Galloway ◽  
Dean A. Glawe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Powdery Mildew ◽  
Bud Burst ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Erysiphe Necator ◽  
Air Samples ◽  
Polymerase Chain ◽  
Species Specific

A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

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