Tag Archives: Pathogen Detection

Response surface methodology to design a selective enrichment broth for rapid detection of Salmonella spp. by SYBR Green Ι real-time PCR

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Appl Microbiol Biotechnol. 2013 Mar 7. [Epub ahead of print]
Zhang Q, Chen T, Yang S, Wang X, Guo H.

Source

Zhejiang Province Key Lab for Food Safety, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, Zhejiang, China, yanyan0014@sina.com.

Abstract

In order to meet dominant growth of Salmonella spp. in a composed system of five pathogens for accurate detection, designing an appropriate selective enrichment broth was clearly needed. First, we built a high-throughput assay procedure based on SYBR Green Ι real-time PCR, which possessed the necessary specificity for Salmonella spp., a good linear standard curve with typical R 2 value (0.9984) and high amplification efficiency (99.0 %). Further, for the larger target biomass in the mixed microflora, acarbose, LiCl and bile salt were selected to optimize their concentrations using response surface methodology (RSM). A central composite design was employed to collect the data and fit the response. A quadratic polynomial model was derived by computer simulation. Statistical analysis was carried out to explore the action and interaction of the variables on the response. In the end, a novel broth (Sal-5) was formulated to allow the efficient enrichment of Salmonella spp. and inhibit the growth of other tested strains. A detection platform was developed, including selective enrichment in Sal-5, DNA extraction by the boiling lysis method and real-time PCR test based on SYBR Green Ι. This work could extend the application of RSM and real-time PCR in the design of other selective enrichment media for common pathogens.

Prevalence of clarithromycin-resistant Helicobacter pylori in patients with chronic tonsillitis by allele-specific scorpion real-time polymerase chain reaction assay

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Laryngoscope. 2013 Feb 12. doi: 10.1002/lary.23777. [Epub ahead of print]
Naserpour Farivar T, Najafipour R, Johari P.

Source

Cell and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.

Abstract

OBJECTIVES/HYPOTHESIS:

To investigate the allelic prevalence of resistance to clarithromycin in the DNA of clinical isolates of Helicobacter pylori obtained from biopsy specimens of patients with chronic tonsillitis by Scorpion real-time polymerase chain reaction (PCR).

STUDY DESIGN:

Pathologic specimens of patients with chronic tonsillitis were used for rapid urease test, and blocks of paraffin-embedded tonsillar tissue were used for McMullen staining, rapid urease test, and Scorpion real-time PCR test.

METHODS:

A total of 103 biopsy samples were obtained from patients with chronic tonsillitis and examined for the presence of clarithromycin resistant H. pylori. Modified McMullen staining and rapid urease test were done on the all the samples. The DNA of specimens was extracted from the pathology blocks, and Scorpion real-time PCR was performed on a final volume of 25 μL.

RESULTS:

Of 103 biopsy specimens, 22 samples were identified as infected by H. pylori, of which none were sensitive to clarithromycin. One had the A2143G genotype, and four had the A2142G genotype. Two had a mixed sensitive and the A2143G genotype, and five had a mixed sensitive and A2142G genotype. One strain had a mixed genotype of sensitive, A2143G, and A2142G.

CONCLUSIONS:

The reported rate of resistance to clarithromycin is of great variation among H. pylori strains isolated from specimens in different countries. Our study showed that the most prevalent genotypes in our H. pylori-positive specimens was A2142G followed by A2143G, which is different from reported results of allele-specific genotyping of H. pylori strains isolated from gastric biopsy and may be a result of cross-resistance to erythromycin and other macrolides.

[New Service] Mycoplasma Detection

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Dear Facility Users,

The Mycoplasma Detection in the cell lines was transferred to the PCR-based detection in the Genotyping Core Facility.

In order to minimize the cost, please prepare your samples as following way,

  1. collect a 1mL sample off of the cells to be tested (do not change medium as it may be needed for retesting the next day)
  2. spin samples at 1500 rpm for 5 minutes and transfer supernatants to new microfuge tubes
  3. label tubes with your name and the name of the cell line
  4. bring samples to the Genotyping Core Facility

If you are interested in the service, please feel free to contact us.

 

A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva

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Lab Chip. 2013 Feb 1. [Epub ahead of print]
Oblath EA, Henley WH, Alarie JP, Ramsey JM.

Source

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. jmramsey@unc.edu.

Abstract

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.

An HPV testing algorithm comprising a combination of the L1 broad-spectrum SPF10 PCR and a novel E6 high-risk Multiplex type-specific genotyping PCR

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J Clin Microbiol. 2013 Jan 30. [Epub ahead of print]
van Alewijk D, Kleter B, Vent M, Delroisse JM, de Koning M, van Doorn LJ, Quint W, Colau B.

Source

DDL Diagnostic Laboratory, Visseringlaan 25, Rijswijk, The Netherlands.

Abstract

HPV epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1 based SPF(10) PCR DEIA LiPA system and a novel E6 based multiplex type-specific system (MPTS123) using Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n=860) and cervical biopsies (n=355) were tested with a focus on HPV detected by the MPTS123 assay (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, -6 and -11). Among the HPV positive samples, identification of individual HPV genotypes was compared. When all MPTS123 targeted genotypes were taken together a good overall agreement was found (κ = 0.801, 95% CI: 0.784-0.818) with identification by SPF(10) LiPA, but significantly more genotypes (P<0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV-16, -35, -39, -45, -58, and -59. An alternative type-specific assay was evaluated, based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed similar results as the expanded MPTS123 Luminex assay.These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs offers a highly accurate method for the analysis of HPV infections and diminishes the rate of false-negative results, which may be particularly useful for epidemiological and vaccine studies.

Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR

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Clin Microbiol Infect. 2013 Jan 24. doi: 10.1111/1469-0691.12153. [Epub ahead of print]
Bergman A, Heimer D, Kondori N, Enroth H.

Source

Department of Clinical Microbiology, Unilabs AB, Skövde, Sweden.

Abstract

The aim of this study was to develop and validate a rapid and sensitive real-time PCR method for detection of all known species of dermatophytes, including identification of Trichophyton rubrum and Trichophyton interdigitale. Fungal DNA was extracted directly from clinical samples by using a pre-lysis step, followed by automated DNA extraction on the MagNA Pure Compact. In total, 202 clinical samples were examined by both conventional culture and by the new PCR method. In 103 (51%) of the samples fungal nucleic acid was detected by PCR, while only 79 (39%) were found to be positive by culture. Out of 103 PCR-positive clinical samples, 94 (91%) were identified as T. rubrum and eight (8%) as T. interdigitale. This real-time PCR is far more sensitive and 2-4 weeks faster than conventional culture for detection of dermatophytes present in clinical samples.

Development of an Efficient Genotyping Method to Detect Obese Mutation in the Mouse Leptin Gene for Use in SPF Barrier Facilities

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J Vet Med Sci. 2012 Dec 10. [Epub ahead of print]
Ayabe H, Ikeda S, Maruyama S, Shioyama S, Kikuchi M, Kawaguchi A, Yamada T, Ikeda T.

Source

Plasma Team, Production Department, CHARLES RIVER LABORATORIES JAPAN, Inc.

Abstract

We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep(ob)) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep(ob)/Lep(ob), Lep(ob)/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.