scholarly journals Detection of Legionella Species in Respiratory Specimens Using PCR with Sequencing Confirmation

2000 ◽  
Vol 38 (5) ◽  
pp. 1709-1712 ◽  
Author(s):  
J. L. Cloud ◽  
K. C. Carroll ◽  
P. Pixton ◽  
M. Erali ◽  
D. R. Hillyard
Keyword(s):  
Legionella Pneumophila ◽  
High Sensitivity ◽  
High Specificity ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Method ◽  
Specific Pcr Assay ◽  
Clinically Significant ◽  
Tract Infections ◽  
Culture Negative

Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive forLegionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionellaspecies by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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.

Development of a Rapid On-Site Method for the Detection of Chicken Meat in Processed Ground Meat Products by Using a Direct Ultrafast PCR System

2020 ◽  
Vol 83 (6) ◽  
pp. 984-990 ◽  
Author(s):  
SUYEON SUL ◽  
MI-JU KIM ◽  
JUNG-MIN LEE ◽  
SUNG-YEON KIM ◽  
HAE-YEONG KIM
Keyword(s):  
Limit Of Detection ◽  
Meat Products ◽  
Accurate Method ◽  
Chicken Meat ◽  
Rrna Gene ◽  
Target Sequence ◽  
Ground Meat ◽  
Specific Pcr ◽  
Pcr Method ◽  
Meat Samples

ABSTRACT In this study, we developed a rapid on-site detection method by using direct ultrafast PCR coupled with a microfluidic chip to identify the presence of chicken meat in processed ground meat products. Chicken-specific PCR primer targeting mitochondrial 16S rRNA gene was newly designed, and its specificity was confirmed against 17 other animal species and 4 different chicken meat samples from different countries of origin. The sensitivity of the chicken-specific ultrafast PCR was 0.1 pg of chicken DNA. To evaluate the limit of detection of the direct ultrafast PCR method, different percentages of chicken meat mixed with pork or beef were prepared. The limit of detection of the direct ultrafast PCR method for the chicken meat–pork and chicken meat–beef mixtures was 0.1% for both raw meat and autoclaved meat. This method was used for 15 commercialized processed ground meat products. In this method, the target sequence was successfully amplified, and the presence of chicken meat in processed ground meat products was identified within approximately 25 min, including the time for sample preparation. Thus, our study shows that this developed direct ultrafast PCR method is a rapid and accurate method for on-site detection of chicken DNA in commercial food products. HIGHLIGHTS

scholarly journals Specific PCR Detection of Tiger, Leopard, and Lion Ingredients from Test Samples

2011 ◽  
Vol 94 (4) ◽  
pp. 1200-1205
Author(s):  
Jijuan Cao ◽  
Junyi Xu ◽  
Ran Liu ◽  
Ke Yu ◽  
Changwen Wang
Keyword(s):  
Food Processing ◽  
Processing Condition ◽  
High Sensitivity ◽  
Animal Protection ◽  
Specific Pcr ◽  
Mitochondrial Cytochrome B ◽  
Inspection And Quarantine ◽  
Powder Samples ◽  
Effect Of Food ◽  
Pcr Method

Abstract A PCR method was developed for specifc detection of tiger, leopard, and lion DNA from test specimens for inspection and quarantine or for law-enforced animal protection. Three pairs of specifc primers were designed based on the mitochondrial cytochrome b gene of tiger, leopard, and lion and used in the PCR testing. To mimic the effect of food processing on the sensitivity of the test, the tiger muscle and bovine bonemeal powder samples were treated at 133°C for 30 min. At this processing condition, the method was sensitive enough to detect as low as 0.05% of tiger-derived ingredients from the mixed bonemeal powders. The data demonstrate that our PCR method is convenient and economic, with high sensitivity and repeatability, and can be used to detect and identify tiger, leopard, and lion ingredients from various test samples.

scholarly journals Title: LAMP Assays for the Simple and Rapid Detection of Clinically Important Urinary Pathogens Including the Detection of Resistance to 3rd Generation Cephalosporins

2021 ◽  
Author(s):  
Lalainasoa Odile RIVOARILALA ◽  
Victor JEANNODA ◽  
Tania CRUCITTI ◽  
Jean Marc COLLARD
Keyword(s):  
Simple Procedure ◽  
High Sensitivity ◽  
High Specificity ◽  
Urine Samples ◽  
Accurate Identification ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Thermal Cycler ◽  
Tract Infections ◽  
Group 1

Abstract Background: Timely and accurate identification of uropathogens and determination of their antimicrobial susceptibility is paramount to the management of urinary tract infections (UTIs). The main objective of this study was to develop an assay using LAMP (Loop mediated isothermal amplification) technology for simple, rapid and sensitive detection of the most common bacteria responsible for UTIs, as well as for the detection of the most prevalent genes (encoding cefotaximases from CTX-M group 1) responsible for resistance to 3rd generation of cephalosporins. Method: We designed primers targeting Proteus mirabilis, while those targeting Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis and the CTX-M group 1 resistance gene were benchmarked from previous studies. The amplification reaction was carried out in a warm water bath for 60 min at 63±0.5 °C. The amplicons were revealed by staining with Sybr Green I. Specificity and sensitivity were determined using reference DNA extracts spiked in sterile urine samples. The analytical performance of the assays was evaluated directly on pellets of urine samples from patients suspected of UTI and compared with culture.Results: We found a high specificity (100%) for LAMP assays targeting the selected bacteria (P. mirabilis, E. coli, K. pneumoniae, E. faecalis) and the CTX-M group 1 when using DNA extracts spiked in urine samples. The sensitivities of the assays were around 1.5 103 Colony Forming Units (CFU) /mL corresponding to the cut-off value used to define bacteriuria or UTIs in patients with symptoms. Out of 161 urine samples tested, using culture as gold standard, we found a sensitivity of the LAMP techniques ranging from 96 to 100 % and specificity from 95 to 100 %.Conclusion: We showed that the LAMP assays were simple and fast. The tests showed high sensitivity and specificity using a simple procedure for DNA extraction. In addition, the assays could be performed without the need of an expensive device such as a thermal cycler. These LAMP assays could be useful as an alternative or a complementary tool to culture reducing the time to diagnosis and guiding for more effective treatment of UTIs but also as a powerful diagnostic tool in resource-limited countries where culture is not available in primary health care structures.

scholarly journals Occurrence of methicillin-resistant strains of Staphylococcus aureus at a goat breeding farm

2009 ◽  
Vol 54 (No. 9) ◽  
pp. 419-426 ◽  
Author(s):  
Z. Stastkova ◽  
S. Karpiskova ◽  
R. Karpiskova
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant ◽  
Specific Pcr ◽  
Type Iv ◽  
Genes Encoding ◽  
Pcr Method ◽  
Specific Pcr Assay ◽  
Mrsa Strains ◽  
Phenotypic Methods ◽  
Species Specific

The aim of this study was to report the detection of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) strains at a veterinary university goat breeding farm and their characteristics. A total of 278 samples collected from animals, milk, environment and farm personnel between June 2006 and March 2008 were examined. The identification of <i>S. aureus</i> isolates was performed by a species specific PCR assay. All detected isolates were tested for resistance to oxacillin and other antimicrobials by phenotypic methods and for the <i>mecA</i> gene by PCR method. Eight MRSA were detected in this study. Five of them originated from goat’s milk and three were recovered from one human carrier of the farm personnel. All obtained MRSA isolates were clonally consistent and were characterized as: <i>SCCmec</i> type IV, spa type t064, seb positive and for genes encoding TSST-1, PVL and exfoliative toxins A and B negative.

scholarly journals Evaluation of real-time PCR for the early detection of Legionella pneumophila DNA in serum samples

2007 ◽  
Vol 56 (1) ◽  
pp. 94-101 ◽  
Author(s):  
Bram M. W. Diederen ◽  
Caroline M. A. de Jong ◽  
Faïçal Marmouk ◽  
Jan A. J. W. Kluytmans ◽  
Marcel F. Peeters ◽  
...  
Keyword(s):  
16S Rrna ◽  
Legionella Pneumophila ◽  
Early Stage ◽  
Pearson Correlation ◽  
5S Rrna ◽  
Rrna Gene ◽  
Serum Samples ◽  
Strong Negative Correlation ◽  
Pcr Assays ◽  
Tract Infections

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=−0.63, P<0.0001). In addition to existing tests for the diagnosis of LD, detection of Legionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.

scholarly journals Development of a triplex real-time PCR for simultaneous detection of allergenic ingredients in processed food

2018 ◽  
Vol 36 (No. 1) ◽  
pp. 22-27 ◽  
Author(s):  
Wenju Zhang ◽  
Yulei Zhao ◽  
Qingjin Xu ◽  
Qin Chen
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Processed Food ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Primer Sets ◽  
Specific Pcr Primer ◽  
Suitable System

SYBR Green real-time or quantitative PCR (Q-PCR) is a suitable system in which to establish a multiplex method to detect allergenic ingredients in food. In this study, a triplex Q-PCR method was developed to detect trace amounts of peanut, soybean and sesame in processed food. Specific PCR primer sets were designed and the concentration of the primers used in the triplex PCR was optimised. The triplex method showed high specificity and sensitivity which were similar to those of the simplex method, and it was applied for the detection of allergenic ingredients in commercially available processed food. The results demonstrate that the developed triplex Q-PCR is a quick, reliable and efficient method for the detection of allergenic ingredients in processed food.

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