Report on Quantitative PCR assay better than traditional culturing methods for assessing the presence

The report is about the “Quantitative PCR assay better than traditional culturing methods for assessing the presence”. A genus Arcobacter involves the different species which are supposed and has the emerging waterborne and the food pathogens. Despite the Arcobacter is connected by the presence of the fecal pollution for the different studies which is also searched through the wastewater prevalence and the isolated species of the Arcobacter butzleri is studies.  In this study the diversity of the genetic is also isolated which is established by the different ERIC-PCR methods and the samples of wastewater are positive for the Arcobacter and it also has the genetic diversity which is observed among the different investigated isolates and it is also belonged by the various ERIC genotypes (Watterworth, et al., 2006). In this report there are different sections discussed like the first section is about the background information, material s and methods and the results section as shown in the below discussion. In this study also develop the highly complex as well as broadly OCR assay for the Arcobacter spp. The changes in the environment that are occurred like the result and it is also driven for changes of ecological in an oral microbiota like the ascendancy of a negative gram of rods and the Actinomyces spp. 

Aim and Justification of Quantitative PCR assay better than traditional culturing methods for assessing the presence

This study aims to establish the prevalence of Arcobacter spp. at the wastewater treatment plant by using the culturing methods. However here culturing method is about the culturing of fish after enrichment and the direct culturing in the wastewater. It also concerns with the direct detection through them-PCR. An aim of this work is to access the presence of arcobacter spp in the water samples taken from the wastewater and other environmental water resources.  The work is also aimed to identify which one culturing methods or the quantitative PCR assay is better. In this study also develop the highly complex as well as broadly OCR assay for the Arcobacter spp. along with it is also developed the assay for the various sources of water. The Arcobacter spp. are the developing pathogens connected by the gastroenteritis in the human? Approximately there are 15 samples which are analyzed through the sequencing of the next generation and it is also collected form 13 shallow of dug well where a deep tube of well along with the river. For the quantification, detection as well as typing the various microbial agents in the different clinical areas along with food safety and veterinary diagnostics (Espy et al , 2006). By understanding the PCR principle with the difficulties by the interoperation as well as presentation data of the limitations of PCR in the various areas of microbial diagnostics as well as the parameters which are very important for the description of PCR performances. The changes in the environment that are occurred like the result and it is also driven for changes of ecological in an oral microbiota like the ascendancy of a negative gram of rods and the Actinomyces spp.  By the molecular techniques which are very advanced like the sequencing as well as cloning of bacterial where it also has a greater proportion of the different species which is presented in the oral cavity and it is also detected through the traditional culture techniques (Levican, 2016) (SCULLION, et al., 2006) .

Background of Quantitative PCR assay better than traditional culturing methods for assessing the presence

A variety of Arcobacter is incorporated together by Campylobacter as well as Helicobacter in a family Campylo-bacteraceae, along with the genera incorporate species which is pathogenic to people along with the creatures. Arcobacter butzleri is a fourth most regular Campylobacter which is isolated like the organism from a stool of human patients through the diarrhea of two free examinations did in Belgium as well as in France. Additionally, in a recent study, the species of Arcobacter that is the fourth common group of the pathogen is also isolated with samples from fecal where persons have the acute enteric disease (Lawson, et al., 2001) (Taylor & Keelan, 2006). In water, the presence of the Arcobacter is also demonstrated which correlates by the fecal pollution presences. In three outbreaks Arcobacter is recovered by drinking water which is also contaminated by different sewages (Noble et al , 2010). The products of foods especially, milk, meat and the selfish which are also determined by contaminated bacteria of the genus with Arcobacter butzleri. The sewage disposal is a critical issue and in modern cities where it is normally delivered and receiving water for wastewater treatment. Then this type of treatment if also reduced degradable organic matter under different controlled conditions where it is also discharged for natural bodies of water. In the utilization of PCR, the great substantial milestone is also introduced monitoring the concept of DNA amplification for the real-time by the fluorescence (Kralik et al, 2017).

Campylo-bacteraceae of Quantitative PCR assay better than traditional culturing methods for assessing the presence

From the family of Campylo-bacteraceae, the genus Arcobacter also involves the various species like the Arcobacter butzleri and the Arcobacter cryaerophilus.  The consideration of an Arcobacter butzleri is also emerging from the waterborne and the potential food of the pathogen. Then there are also different Arcobacter butzleri. Species and the Arcobacter Cryaerophilus which is connected by diarrhea as well as mastitis in various animals. The pathogenic, as well as the epidemiology, has the role of Arcobacter butzleri in the human disease which is still unknown and it is also presented by the different bacteria of the microbiota of the seawater.  The investigation which is concluded by the water and it is might play a significant role in the microbe transmission of animals and the human. Then for the Arcobacter butzleri which is recorded for in the most groundwater which is contaminated, and well is provided by portable water.  There is nothing unknown potential relationship among the several indicators along with the Arcobacter presences. Thus there is a different study which is also determined by the prevalence of the Arcobacter in the environmental species where the water is also established by the relation of the fecal contaminations (Postollec et al , 2011).  In this study, the stream is the fecally which also has the contaminated freshwater and the discharging of the various beaches along with the sampled is selected by the Arcobacter butzleri quantification along with the sampling which is formed in three times and the stream before seashore and point of entrance in the freshwater of the sea. The connection among the Arcobacter spp. as well as the indicators of the bacterial which has fecal pollution and it is also revealed by different samples of various origins (Houf, et al., 2001; Webb, et al., 2016).  

Methods and experimental design of Quantitative PCR assay better than traditional culturing methods for assessing the presence
Water processing and Samples

 Samples of the wastewater which is collected by the different six-date of samples at the wastewater treatment plant. In the affluent and the influent water, there are different sampling points for the secondary and the primary tanks of sedimentations in the bioreactor tank. Then in this, there are 2-lire sterile polypropylene samples is collected by bottles and then it's chilled by the ice of transport where the microbiological assays are started on the same day of sampling. The filter which is introduced for the tube containing the 9mL of Arcobacter and the supplemented enrichment of the broth by the CAT supplement antibiotic. Seawater Aliquots, as well as little plus enormous microscopic fish, were straightforwardly immunized into the proper culture media. Enormous microscopic fish are the food of small and some large size fishes which includes tuna fish and polar fish. These enormous microscopic fish has microscopic bodies that feed on the anchovies fish and provide prey to the shark and fish bigger than megalodon. Key examples of enormous microscopic fish contains sunfish, anchovy, piranha, and candiru. Before vaccination, tests of huge microscopic fish were normalized for 1 min in a glass homogenizer at 5,800 pm. All examples were vaccinated into Arcobacter stock CM965 (Oxoid) enhanced with cefoperazone, amphotericin B, teicoplanin specific enhancement SR 174E, which was particular for the development of Arcobacter species, along with cefoperazone, amphotericin B specific enhancement SR 155 for particular improvement of Arcobacter butzleri. The detection of molecules of water has the 2 tubes and it is centrifuged and obtained by the pellet which is resuspeneded as well as washed by 3 times (Fera et al , 2004).

Culturing Procedure of Quantitative PCR assay better than traditional culturing methods for assessing the presence

By the culturing procedure of the direct detection of the 200 water is no enriched by the tube and it is transferred through the face of the 0.45 membrane filter which is also placed by the blood agar medium. After enrichment, the culturing is the 200 water of the incubated of Arcobacter where the enrichment is also transferred on the face of bran filter and it’s placed through the agar medium which is also allowed by the serious filter. The filter is removed along with the plates and it is also incubated under various conditions. The post-enrichment as well as the direct culturing which is processed for the duplicate order which is permits parallel incubation of the microaerophilic along with the aerobic conditions. The microaerophilic condition which is generated through the use of sachets of container systems (Olwagen et al , 2017).

Counting of Arcobacter of Quantitative PCR assay better than traditional culturing methods for assessing the presence

The counting of the direct Arcobacter is also carried through wastewater samples and it is also collected by previously. The samples of the brief water which are tenfold of diluted 0.1% with the peptone water and every tenfold of the dilution is also inoculated by selective Arcobacter and the agar plate selective isolation is supplemented by the 100mg liter. The dilution of peptone in the water is also used to enumerate the Arcobacter by using the method of MPN (Yang et al, 2003).

Expected results and impact of Quantitative PCR assay better than traditional culturing methods for assessing the presence

From the 29 to 30 samples approximately 96.7%  the Arcobacter spp. which is recovered and it is also isolated through the culturing which is confirmed and it belongs to a genus of the Arcobacter as shown in table  1in the below appendix 1. All these isolates are also genotyped by the ERIC-PCR along with the various patterns which are presented and it is also belonged by the 424 different genotypes, and then the global diversity of genetics is also 65.1% which is shown in below table 1. The previous study which is also used by various culture of media and the prevalence of protocols by the Arcobacter spp. form the samples of the wastewater which is ranged the 40 to 100%. Then there is the method of genotyping which is also applied by the high diversity of the gene which is observed and a report of 90.2% of isolates is also belonged by the different PCR genotypes. The diversity of the high genetic which is previously explained through the possible different sources of the containment like the consequences of the rearrangements of a genomic (Massó et al , 2019).

By using the 30 samples which are studied the 28 is positive through directed plating of 29 through the post-enrichments through PCR which is presented by the various samples is taken from the wastewater treatment plant is negative through three methods and Relative costs of various SARS PCR assays in table  2. For this study the ethic approval for health and safety is shown in the below appendix 2

References of Quantitative PCR assay better than traditional culturing methods for assessing the presence

1.      Collado et al, L., 2008. Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution. Environmental Microbiology, 10(6), p. 1635–1640.

2.      Espy et al , M. J., 2006. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clinical Microbiology Reviews, 19(9), p. 165–256.

3.      Fera et al , M. T., 2004. Detection of Arcobacter spp. in the Coastal Environment of the Mediterranean Sea. Applied and Environmental Microbiology, 7(3), p. Applied and Environmental Microbiology.

4.      Houf, K. et al., 2001. Development of a new protocol for the isolation and quantification of Arcobacter species from poultry products. International Journal of Food Microbiology, 71(2-3), pp. 189-196.

5.      Kralik et al, P., 2017. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Frontiers in Microbiology.

6.      Lawson, A. J., On, S. L. W., Logan, J. M. J. & Stanley, J., 2001. Campylobacter hominis sp. nov., from the human gastrointestinal tract. International Journal of Systematic and Evolutionary Microbiology, 51(2), pp. 651-660.

7.      Levican, A., 2016. The Use of Two Culturing Methods in Parallel Reveals a High Prevalence and Diversity ofArcobacterspp. in a Wastewater Treatment Plant. BioMed Research International, p. 1–9.

8.      Massó et al , N. S., 2019. The Use of a DNA-Intercalating Dye for Quantitative Detection of Viable Arcobacter spp. Cells (v-qPCR) in Shellfish. Frontiers in Microbiology.

9.      Noble et al , R. T., 2010. Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, p. 437–7443.

10.  Olwagen et al , C. P., 2017. Comparison of traditional culture and molecular qPCR for detection of simultaneous carriage of multiple pneumococcal serotypes in African children. Scientific Reports, 7(1).

11.  Postollec et al , F., 2011. Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiology, 28(5), pp. 848-61.

12.  SCULLION, R., HARRINGTON, C. S. & MADDEN, R. H., 2006. Prevalence of Arcobacter spp. in Raw Milk and Retail Raw Meats in Northern Ireland. Journal of Food Protection, 69(8), pp. 1986-1990.

13.  Taylor, D. E. & Keelan, M., 2006. Campylobacter and Arcobacter spp.. s.l.:Principles and Practice of Clinical Bacteriology, Second Edition. John Wiley & Sons, Ltd.

14.  Watterworth, L. et al., 2006. Survival of various ERIC-genotypes of Shiga toxin-producing Escherichia coli in well water.. Water, air, and soil pollution, 177(1-4), pp. 367-382.

15.  Webb, A. L. et al., 2016. Comparative Detection and Quantification of Arcobacter butzleri in Stools from Diarrheic and Nondiarrheic People in Southwestern Alberta, Canada. Journal of clinical microbiology, 54(4), pp. 1082-1088.

16.  Yang et al, C., 2003. Application of real-time PCR for quantitative detection ofCampylobacter jejuniin poultry, milk and environmental water. FEMS Immunology & Medical Microbiology, 38(3), p. 265–271.