3,3′-Thiodipropionic acid (TDP), a possible precursor for the synthesis of polythioesters: identification of TDP transport proteins in Variovorax paradoxus TBEA6

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11294-y ◽

2021 ◽

Author(s):

M. Venkateswar Reddy ◽

Alexander Steinbüchel

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Significant Shift ◽

Wild Type ◽

The Real ◽

E Coli ◽

Variovorax Paradoxus ◽

Protein Ligand Interactions ◽

Mutant Strains ◽

Ligand Interactions

Abstract3,3′-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

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Rumen Microbiome Composition Determined Using Two Nutritional Models of Subacute Ruminal Acidosis

Applied and Environmental Microbiology ◽

10.1128/aem.00739-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7115-7124 ◽

Cited By ~ 283

Author(s):

Ehsan Khafipour ◽

Shucong Li ◽

Jan C. Plaizier ◽

Denis O. Krause

Keyword(s):

Lactic Acid ◽

Dairy Cattle ◽

Real Time ◽

Real Time Pcr ◽

Rrna Genes ◽

Subacute Ruminal Acidosis ◽

The Real ◽

E Coli ◽

Ruminal Acidosis ◽

Rumen Microbiome

ABSTRACT Subacute ruminal acidosis (SARA) is a metabolic disease in dairy cattle that occurs during early and mid-lactation and has traditionally been characterized by low rumen pH, but lactic acid does not accumulate as in acute lactic acid acidosis. It is hypothesized that factors such as increased gut permeability, bacterial lipopolysaccharides, and inflammatory responses may have a role in the etiology of SARA. However, little is known about the nature of the rumen microbiome during SARA. In this study, we analyzed the microbiome of 64 rumen samples taken from eight lactating Holstein dairy cattle using terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA genes and real-time PCR. We used rumen samples from two published experiments in which SARA had been induced with either grain or alfalfa pellets. The results of TRFLP analysis indicated that the most predominant shift during SARA was a decline in gram-negative Bacteroidetes organisms. However, the proportion of Bacteroidetes organisms was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA (35.4% versus 26.0% and 16.6%, respectively). This shift was also evident from the real-time PCR data for Prevotella albensis, Prevotella brevis, and Prevotella ruminicola, which are members of the Bacteroidetes. The real-time PCR data also indicated that severe grain-induced SARA was dominated by Streptococcus bovis and Escherichia coli, whereas mild grain-induced SARA was dominated by Megasphaera elsdenii and alfalfa pellet-induced SARA was dominated by P. albensis. Using discriminant analysis, the severity of SARA and degree of inflammation were highly correlated with the abundance of E. coli and not with lipopolysaccharide in the rumen. We thus suspect that E. coli may be a contributing factor in disease onset.

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Effects of rainfall on the occurrence of human adenoviruses, total coliforms, and Escherichia coli in seawater

Water Science & Technology ◽

10.2166/wst.2006.473 ◽

2006 ◽

Vol 54 (3) ◽

pp. 225-230 ◽

Cited By ~ 22

Author(s):

E. Haramoto ◽

H. Katayama ◽

K. Oguma ◽

Y. Koibuchi ◽

H. Furumai ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Tokyo Bay ◽

Rainfall Events ◽

Total Coliforms ◽

The Real ◽

E Coli ◽

Human Adenoviruses ◽

Seawater Samples

A two-month survey was conducted in order to evaluate the effects of rainfall on the fate of microorganisms in seawater in the Tokyo Bay, Japan. The seawater sample (1,000 mL) was applied to a method to concentrate virus, followed by a quantification of human adenoviruses using the real-time PCR. Total coliforms and E. coli, which were determined by the colony forming method, were detected in all 47 seawater samples, while human adenoviruses were detected in 38 (81%) of the samples. The concentration of tested microorganisms showed 1–2 log units increase after rainfall events, followed by the gradual decrease to the level before the rainfall within a few days.

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Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1366 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1366-1372 ◽

Cited By ~ 104

Author(s):

LUXIN WANG ◽

YONG LI ◽

AZLIN MUSTAPHA

Keyword(s):

Escherichia Coli ◽

Detection Limit ◽

Real Time ◽

Real Time Pcr ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Quantitative Detection ◽

Escherichia Coli O157 ◽

The Real ◽

E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

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Brucella Melitensis invA Gene (BME_RS01060) Transcription Is Promoted Under Acidic Stress Conditions.

10.21203/rs.3.rs-516691/v1 ◽

2021 ◽

Author(s):

Raúl Sauceda-Becerra ◽

Hugo Barrios-García ◽

Julio Martínez-Burnes ◽

Beatriz Arellano-Reynoso ◽

Alejandro Benítez-Guzmán ◽

...

Keyword(s):

Real Time ◽

Gene Transcription ◽

Real Time Pcr ◽

Brucella Melitensis ◽

Survival Rates ◽

Time Dependent ◽

Acidic Stress ◽

Wild Type ◽

Mutant Strains ◽

Acidic Conditions

Abstract The invA gene of Brucella melitensis codes for a NUDIX (nucleoside diphosphate linked to moiety X) hydrolase related to invasiveness. The objective of this work was to evaluate invA transcription under acidic conditions. The invA gene transcription was up regulated at pH 3 and pH 5 observed with semiquantitative real-time PCR in B. melitensis 133 strain. Results indicated that invA gene transcription at pH 3 showed a basal and decreased transcription compared to that of pH 5 incubation. Transcription levels of the dnaK gene were similar to those obtained with invA gene. The survival rates of wild type and invA mutant strains at pH 5 were above 90% in all post-incubation times. In contrast, at pH 3 there was a time-dependent reduction on both strains at 15 min (P < 0.05). These results suggest that invA gene transcription is promoted under acidic conditions in Brucella melitensis.

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DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2019.3.8 ◽

2019 ◽

pp. 60-66

Author(s):

Viet Quynh Tram Ngo ◽

Thi Ti Na Nguyen ◽

Hoang Bach Nguyen ◽

Thi Tuyet Ngoc Tran ◽

Thi Nam Lien Nguyen ◽

...

Keyword(s):

Bacterial Meningitis ◽

Real Time ◽

Real Time Pcr ◽

Diagnostic Methods ◽

Type B ◽

E Coli ◽

Pcr Assays ◽

Set Up ◽

Etiological Agents ◽

Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

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APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2017.3.15 ◽

2017 ◽

pp. 99-103

Author(s):

Van Bao Thang Phan ◽

Hoang Bach Nguyen ◽

Van Thanh Nguyen ◽

Thi Nhu Hoa Tran ◽

Viet Quynh Tram Ngo

Keyword(s):

Cervical Cancer ◽

High Risk ◽

Real Time ◽

Real Time Pcr ◽

Dot Blot ◽

The Real ◽

Dot Blot Assay ◽

Hpv Types ◽

High Risk Hpv ◽

Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

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Real-Time PCR as an Alternative Technique for Detection of Dermatophytes in Cattle Herds

Animals ◽

10.3390/ani11061662 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1662

Author(s):

Dominik Łagowski ◽

Sebastian Gnat ◽

Aneta Nowakiewicz ◽

Aleksandra Trościańczyk

Keyword(s):

Light Microscopy ◽

Real Time ◽

Real Time Pcr ◽

Direct Analysis ◽

Carrier Status ◽

The Real ◽

Detection Techniques ◽

Direct Light ◽

Microscopy Analysis ◽

Cattle Herds

Dermatophytes are filamentous fungi with the ability to digest and grow on keratinized substrates. The ongoing improvements in fungal detection techniques give new scope for clinical implementations in laboratories and veterinary clinics, including the monitoring of the disease and carrier status. The technologically advanced methods for dermatophyte detection include molecular methods based on PCR. In this context, the aim of this study was to carry out tests on the occurrence of dermatophytes in cattle herds using qPCR methods and a comparative analysis with conventional methods. Each sample collected from ringworm cases and from asymptomatic cattle was divided into three parts and subjected to the real-time PCR technique, direct light microscopy analysis, and culture-based methods. The use of the real-time PCR technique with pan-dermatophyte primers detected the presence of dermatophytes in the sample with a 10.84% (45% vs. 34.17%) higher efficiency than direct analysis with light microscopy. Moreover, a dermatophyte culture was obtained from all samples with a positive qPCR result. In conclusion, it seems that this method can be used with success to detect dermatophytes and monitor cowsheds in ringworm cases and carriers in cattle.

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EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains

Animal Biotechnology ◽

10.1080/10495398.2016.1268620 ◽

2017 ◽

Vol 28 (4) ◽

pp. 248-252 ◽

Cited By ~ 1

Author(s):

Sachin S. Pawar ◽

Chetan D. Meshram ◽

Niraj K. Singh ◽

Mohini Saini ◽

B. P. Mishra ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bovine Herpesvirus ◽

Wild Type ◽

Pcr Assay ◽

Bovine Herpesvirus 1 ◽

Glycoprotein E ◽

Herpesvirus 1

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Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-297 ◽

2012 ◽

Vol 75 (4) ◽

pp. 743-747 ◽

Cited By ~ 23

Author(s):

BWALYA LUNGU ◽

W. DOUGLAS WALTMAN ◽

ROY D. BERGHAUS ◽

CHARLES L. HOFACRE

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Salmonella Enteritidis ◽

Culture Method ◽

Pcr Assay ◽

Culture Methods ◽

The Real ◽

Selective Enrichment ◽

Conventional Culture ◽

Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

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