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The author has introduced the topic about detection of campylobacter coli, C.jejuni, and salmonella enterica on poultry carcasses by using PCR and ELISA techniques. He has explained that Poultry carcasses also one of the source for microorganisms which I have mentioned above then he also explained that how to identify these microorganisms by using molecular techniques and how it cause diseases to human being. Both campylobacter and Salmonella infectious to human. Salmonella bacteria generally cause a self limiting gastroenteritis in healthy adults and rarely fatal bacteremia in the very young and elderly ones. Campylobacter coli and campylobacter jejuni most human infectious cause a debilitating neurological disorder, Guillain Barre Syndrome.
Conventional cultural methods are very long process to detect salmonella and campylobacter species because it needs enrichment in selective broth, followed by isolation on selective differential agar. Micro aerobic condition is essential to grow campylobacter spp. which makes the task of isolation laborious and costly. In case of salmonella enterica primary and secondary enrichment culture are required to isolate from food. Its labor intensive and expensive when large number of samples must be processed. Here there is no sensitive non cultural detection method for these food borne micro-organisms.
Therefore molecular techniques such as PCR (polymerase chain reaction) ELISA (enzyme linked immunosorbent assay) have proven to be specific and sensitive methods for identifying infectious pathogens. In PCR technique direct identification of organisms without prior isolation and purification from samples but PCR-ELISA is more sensitive than conventional gel-based PCR. Because PCR-ELISA involves incorporation of chemically tagged nucleotides into the PCR amplicon and subsequent detection of the PCR product with antibody enzyme conjugate that recognizes the unique chemical label present in the incorporated nucleotides. In this study specific multiplex PC primer set and probes based on the salmonella virulence gene invA and the campylobacter ceu E gene, which encodes a lipoprotein involved in siderophore transport for PCR-ELISA to screen poultry carcasses for these two food-borne pathogens. PCR- polymerase chain reaction is defined as a molecular technique which allows the production of large quantities of a specific DNA from a DNA template using a simple enzymatic reaction without a living organism(http://www.pcrstation.com/definition-of-pcr/) PCR-ELISA-also known as PCR ELOSA are a capture a assay for nucleic acids that mimic enzyme linked imunosorbant assays. In this assay, PCR products hybridized to an immobilized capture probe. The assay thus measures sequences internal to the PCR product and is a less expensive assay and an alternative to real time PCR(http://www.btc-bti.com/pcrelisa.htm)
Setting up and testing hypothesis is an essential part of statistical inference. In order to formulate such a test, usually some theory has been put forward, either because it is believed to be true or because it is to be used as a basis for argument, but has not been proved.
Null Hypothesis: It is represented by H0 a theory that has been put forward either because it is believed to be true or because it is to be used as a basis for argument but has been proved. The final conclusion once the test has been carried out is always given in terms of the null hypothesis. We either "rejects H0 in favor of H1 we never conclude "reject H1" this concept introduced by R.A fisher. Weisstein Eric W. it is rejected due to (p<0.05) and so there is no significant different between the values.
Alternative Hypothesis: It is represented by H1. Is a statement of what a statistical hypothesis test is set up to establish. The final conclusion once the test has been carried out is always given in terms of the null hypothesis. We either "reject H0 in favor of H1". This rejects the null hypothesis because (p<0.05) at 95% confidence level so, there is no significant difference between the values obtained for three vales.
Two errors we can make out in hypothesis test a type I error occurs when the null hypothesis is reected when it is fact true, that is Ho is wrongly rejected, type II would occur if it was concluded that the two values produced the same effect. I.e. there is no difference between the two values on average, when in fact they produced different ones.( http://www.stats.gla.ac.uk/steps/glossary/hypothesis_testing.html)
Materials and methods:
Isolation of salmonella and campylobacter from and poultry environment and detecting C.jejuni, C.coli and Salmonella enterica.
Sealed chicken carcasses rinsed by using sterile distilled water and cultured for campylobacter. Rinsed water serially diluted (1:10) and one tenth of culture spread on each campy-cefex agar plate with sterile plastic then inoculating loop and plates were incubated at 420C for about 36 to 48 hrs in a microaerobic condition (O2 5%,CO2 10%, N285%). Conducting microscopic examination and colony morphology each sample was confirmed campylobacter genus. By latex agglutination test kit identify C.jejuni or C.coli . For salmonella identification is by taking samples broiler houses using drap swabs and pad soaked with double strength skim milk placed in 100ml of tetrathionate brilliant green broth (TBG) incubated at 41.50C for about 18hrs. carcasses rinsed with 250ml of buffered peptone and incubated TBG at 410C for 18hrs streaked onto an X2T4-BG biplate and incubate for 370C .Which colony was produce H2 gas on X2T4 plates were identified as salmonella by salmonella specific antiserum. The followed by PCR-ELISA molecular technique by using target gene invA(salmonella) invA and invar capture probe for campylobacter ceuE9Camylobater) jcF and jcR2 resectively.
In this study for isolation of salmonella and campylobacter species serial dilution have been used. The advantage of serial dilution is reduce concentration and increases the specificity. Serial dilution is made by making the same dilution step over and over using the previous as the input to the next dilution in each step. Since the dilution-fold is the same in each step, the dilutions are geometric series. (http://www.bio.umass.edu/micro/immunology/elisa/serial.html)
To detect Campylobacter species and salmonella species PCR-ELISA molecular technique used here this is a rapid and cost-effective as well. For extraction of DNA extraction Mo Bio DNA purification and isolation kit used by this kit we can process up to 20 samples simultaneously and it reduce the need to handle waste during nucleic acid purification.(http://www.mobio.com/lab-supplies/powervac-manifold-mini-system.html) Hastings software used here to search for PCR primers specific for C.jejuni.
Kappa test: Cohen's kappa is a measure of association (correlation or reliability) between two measurements of the same individual when the measurements are categorical. It is used to study the agreement of two raters. Each rater classifies each individual into one of k categories. The statistically significant kappa test indicates that we should reject the null hypothesis that the ratings are independent (i.e. kappa=0) Rules of thumb for kappa: values less than 0.40 indicate low association; values between 0.40 and 0.75 indicate medium association, and values greater than 0.75 indicate high association between the two raters. (http://rimarcik.com/en/navigator/z-nominal.html)
Chi-square test: According to R.A.Fisher and F.Yates Chi-square is a statistical test commonly to compare observed data with data we would expect to obtain according to a specific hypothesis. This states that there is no significant difference between the expected and observed result. The chi-square test indicated a significant correlation between PCR-ELISA and culture methods for detecting salmonella (p<0.001), while there was good agreement according to kappa test (0.63).
The PCR-ELISA values for OD(optical density) at 405nm for negative control strains (OD 405 range 0.162 to 0.235) were recorded and served as the cut off point for identifying position. Any reaction with OD405 between 0.22 and 0.26 was judged as weakly positive greater 0.26 were considered strongly positive. In this study without pre-enrichment step we expect with PCR-ELISA 31 of 32 samples were PCR positive for campylobacter among these there were 4 weak positive and 27 strong positive there was one false.
The statistical methods which I have mentioned above are relevant and Cytel software was used in this research paper. It is one of the best software to do statistical analysis.
Intellectual property rights (IPR)
It is defined as the taking the rights on a creative effort of the individual. It is divided into seven sub branches under the TRIPS (trade related aspects of intellectual property rights. They are as follows.
Patent is to provide legal rights and protection for person's creative plans who makes the new concepts and secure from unauthorized so in future it will bring lots of good thoughts about PCR technique. Therefore there is no point to give patent here because this procedure has creative new ideas.
Health and safety
For health and safety we should give first preference while carrying out this research proper safety and health measures should be taken into consideration because here we were handled food borne pathogens.
Quality control applied in this procedure was good it h
This research has got better and accurate results for conducting relevant molecular techniques using related probes and primers to detected campylobacter and salmonella species and economical more beneficial time saving procedure. PCR-ELISA detection scheme in this study valuable tool in screening large number of samples for taking 7 hour to perform cost just 3 per sample this method is easier than competitive PCR methods developed to detect specific DNA without gel electrophoresis, ethidium bromide staining and UV detection of the amplicon, southern hybridization of the PCR product wih probes and the detection of the hybrids with specialized equipments. Another advantage of the PCR-ELISA compared to convential PCR. Particularly for private sector requiring simultaneous large-scale sample screenings.