Using a Real-Time PCR for Diagnosis in the Laboratory of Human Infection with a Whooping Cough - Essay Example

MOLECULAR BIOLOGY al Affiliation) Table of Contents Table of Contents 2 Problem to be addressed. 3 Introduction 3 Justification of the Technique 4 Preliminary Protocol 5 Protocol for the Technique Optimization 6 Materials 6 Apparatus 6 Reagents 7 Controls 7 Procedure 7 Preparation of reagents 8 Pre-PCR 8 Results 8 Interpretation of the results 9 Kits 9 Suggestion for Approaches 10 Final SOP Suggestion 11 List of Suppliers and Costing 11 Assessment 13 Reference 16 Key words: Protocol, Risk Assessment Problem to be addressed. This project involves using a real time PCR for diagnosis in the laboratory of human infection with a whooping cough. Introduction Bordetella pertussis causes a whooping cough; a bacterial infection that infects the respiratory. The infection is characterized by post tussive vomiting, paroxysmal cough, and whoop. The most severe infection affects the young children and the infants. In adults and adolescents, it affects those constitute the reservoir and are caused from the infections that spread to younger children. The disease is normally mild. Pertussis has remained to be an endemic in the entire world and is considered to A clear understanding of the biological functions of the recently be a disease that is cyclic, peaking after 3 to 5 years. From the year 2011, the cases of Pertuhas have been rising in various parts of the world. Also in regions having sustained coverage of high vaccination (Pestana, 2010). In European Countries, the situation is the same, with various nations observing a rise in adults, adolescents, and infants. ECDC is taking note of the improvement and harmonization of the diagnosis for the outbreak detection, monitoring, and outbreak to assure the comparability and quality of the data. In 2011, ECDC came up with a project coordination of tasks for the surveillance of the lab of the whooping cough in EEA and Member nations. One of the reasons behind the project was to come up with a document on the guidance that related to using real time PCR on the extracted DNA from the specimens found in clinics gotten from the clients that have whooping cough. The protocol and guidance are meant for the PCR on the extracted DNA from the specimens that are suspected of contracting the whooping cough. The infection behind this is the B. parapertussis or even B. pertussis. The suitable samples are either NPS (nasopharyngeal swabs or NPA (nasopharyngeal aspirates) which were used in sampling the specimens from the patient’s posterior nasopharynx (Pestana, 2010). Other species of Bordetella like B. brondhiseptica and B.holmesii can be detected using the PCR from the extracted DNA from nasopharyngeal or nasopharyngeal swabs, and various amplification goals that were used in Bordetella are found in more than single species of Bordetella. It is crucial that the results of PCR are well interpreted. Justification of the Technique Pertussis’ clinical symptoms are typical in children who are not vaccinated. The symptoms include vomiting, coughing spams, and whooping. However, in scenarios of unvaccinated infants and neonates normally present with apnea as a symptoms. The B.pertussis infections in adults, adolescents, and older children represents contacts with the antigens, therefore symptoms tend to differ widely (“Detecting PCR pathogens in real time”, 2005).  They are normally atypical and only shows as coughing that prolongs without any other typical symptoms. Subsequently, the clinical diagnosis need to be justifies using a laboratory experiment. When the sensitivity of PCR reduces after a prolonged period of symptom, it is crucial to carry out PCR sampling during the beginning phases of the disease. Is a person experiences the symptoms more than a month, IgG-anti PT test, normally referred to as serology is carried out. Additionally, PCR from the cases that are secondary are recommended because the disease is one of the highly contagious. It is proper to sample PCR justification by collecting nasopharyngeal aspirate or nasopharyngeal swab. Either anterior nasal swabs or throat swabs should not be recommended because the bacteria of B.pertussis are mainly attached to ciliated epithelium found in the nasopharynx. The advised procedures to swab are sample through the nose with the help of the thin flexible swab. Normally swabs that are made from rayon or Dacron can be used (“Detecting PCR pathogens in real time”, 2005). Those swabs made of calcium alginate or cotton is not good with the PCR techniques. The wire swab that is twisted and flexible is normally used and can be advised. Gloves need to be used to prevent the issues of contamination. An illustration of the suitable swab’s transport media are Amie’s medium with Stuart has and charcoal medium. When using the swabs in transport media that are liquid in nature, it is important to avoid contaminating swab thaft because the contamination easily is transferred to medium. This is because some of the pertussis vaccines have B. pertussis DNA, it is advised that specimen sampling and vaccination be performed in various rooms. Various commercial kits are found for the extraction of DNA. To be specific, the Qiagen (QIAamp DNA mini kit) and highly pure PCR template Roche are normally used. The extractions with the help of the Roche or Chelex are proper options (Dorak, 2006). Preliminary Protocol The protocol and the guidance are intended for using the real time PCR on DNA that was gotten from the clinical samples obtained from the patients who are suspected of having whooping cough or those infected with the nasopharyngeal and sampled from posterior nasopharynx. The protocol resides on the Roche, since most of laboratories are using the conventional LightCycler for detecting the B.paraperussis and B.pertussis and the researched guidelines protocol concentrated on LightCycler. Although there are various varied selection for machines of PCR. It was crucial to choose one to come up with a proper protocol. However, the protocol is important as basis to design an in-house technique for other formats (McPherson & Møller, 2006). Protocol for the Technique Optimization The details for PCR are presented in three varied targets. Each of the PCR need to run separately. As shown in the table below IS481 Number target of high copy found in B. holmessi, Pertussis, and B. bronchiseptica IS1001 Number target of high copy found in B.brobchiseptica, B.paraoertussis, and B.holmessi ptxA-Pr Number target of single copy found in Pertussis, B.bronchiseptica, and B.parapertussis, and B.pertussis specific assays that target the region among the most well characterized. Materials The sample material involves the aspirates or the Nasopharyngeal from patients who are suspected to be infected with B.parapertussis or B.pertussis. Apparatus The Micro centrifuge Pipettes Pre-sterilized pipette tip which are resistant to aerosol Aerosol resistant that is pre-sterilized LightCycler tools using the capillaries LightCycler Capillaries, Adapters, and capillary related tools. Reagents PCR grade water Uracil DNA Glycosylate LightCycler®FastStart and MgCl2 LightCycler®FastStart (IS1001) Applied Bio systems. Controls It is advised that the Bordetella DNA sample be included as one of the positive control. For ptxA-Pr and IS481, this needs to be the B.pertussis. For the IS1001 need to be B.parapertussis. For negative control, it is advised that PCR grade water sample need to be one of the negative control (Edwards, 2004). For the internal process control, it is advised that the internal process control need to be included. To prevent contamination, the synthetic deoxynucleotide is employed rather that the dTTP in the mixture of PCR, enabling UNG to be used before the new assay to prevent the amplicon’s carry over. UNG catalyzes uracil removal from the DNA that is double and single stranded and synthesized in the dUTP presence. Consequently, every PCR protocol, two probes and primers are needed (Logan, Edwards & Saunders, 2009). Procedure The extraction of DNA from the nasopharyngeal sample can be conducted manually with the help of the Qiagen, Chelex or Roche. An automated technique like the MagNa Pure Compact is also an option. Preparation the dilution in the ratio of 1:10 of every DNA in the PCR grade water. Each of the sampled DNA needs to be assessed in the dilution and undiluted form to predict the potential inhibition. Preparation of reagents Following the guidance in the kit insert the LightCycler® components and mix the contents accordingly. The master mix is prepared in a sterile tube through multiplication of the mix by the cycled reaction (Lázaro, 2013). Every DNA sample is assessed twice in undiluted form. Room for both negative and positive control is important. The master mix volume is shown below. Pre-PCR The cooling block of the LightCycler having, the centrifuge adapters and reagent’s room need to be stored at 2 to 8 degrees. The master mix is place in the reagents section and mounts the number of capillaries inside the adapters. Then the master mix is pipetted into the cooled capillaries. Add DNA samples to the capillary. For the –ve control, PCR grade water pipettes into the capillary. For the +ve control, B.pertussis pipettes into the capillary. Close every capillary having a stopper and keep the adapters having the capillary in a centrifuge. Then place the rotor’s capillary of LightCycler instrument. Results Interpretation of the results The interpretation of the results from the 3 different PCR runs resides on the combination of both negative and positive results in three different analyses. IS481 ptxApr IS1001 Outcome + - - Bordetella positive (B.holmesii, B. parapertussis, B. bronchiseptica) - + - Equivocal + + - B.pertussis +ve - - + B. bronchiseptica, B.parapertussis + + + Co-infection with various Bordetella spp. + - + Co-infection with various Bordetella spp. - - - Negative Kits The Real-time PCR tools enable simultaneous multiple target detections since they have changing excitations of 6 or more varied detection of colors. Various apparatus are present for the real time PCR. Among those that are normally used involve Applied Bio systems, Light Cycler, I Cycler, and Smart Cycler. All the tools are taken to be important. The process of selecting the real time PCR benchmark is dependent on the application. Some of the instruments are created for lower capacity and other are designed for applications that are high throughput. The 1st generation Roche used the capillary tubes that have thin walled tubes (“Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications”, 2010). Presently, thin instruments and tools use the standard plates like the 96 well plates. The real time PCRs normally maximize the small reaction volume like 20 to 25 microliters. Various commercial kits are present for the Bordetella parapertussis and Bordetella pertussis detection in specimens in clinics. From the kits, the Focus Diagnostics, Cepheid, and Argene have been assessed and proven suitable. Conversely, the Shanghai ZJ Biotech was evaluated and found to be very unsuitable. The new kits of PCR for the Bordetella parapertussis and Bordetella pertussis normally introduced to the market. Generally, the target amplification and results interpretation need to abide by the guidance below based on the amplification target choice. Suggestion for Approaches The laboratory diagnosis of the disease is not easy due to sensitivity and specifies variation of the various methods. The difference in the protocols and methods among the countries found in Europe has been flagged out (Mackay, 2007). ECDC is taking note of the improvement and harmonization of the diagnosis for the outbreak detection, monitoring, and outbreak to assure the comparability and quality of the data. In 2011, ECDC came up with a project coordination of tasks for the surveillance of the lab of the whooping cough in EEA and Member nations. One of the reasons behind the project was to come up with a document on the guidance that related to using real time PCR on the extracted DNA from the specimens found in clinics gotten from the clients that have whooping cough. Final SOP Suggestion Various targets of amplification are possible the diagnostics of Bordetella PCR. However, majority of the targets are found in multiple species of Bordetella, and majority of the target combination will be unable to identify one species of Bordetella. The multiplex or the sequential PCR are important for it to identify the organisms that are infecting. Conversely, employing the multiplex PCR than the singleplex can minimize the analytical sensitivity. List of Suppliers and Costing Some of the frequently used targets of amplification for the Bordetella related PCR are outlined in the table below. IS481 Number target of high copy found in B. holmessi, Pertussis, and B. bronchiseptica IS1001 Number target of high copy found in B.brobchiseptica, B.paraoertussis, and B.holmessi ptxA-Pr Number target of single copy found in Pertussis, B.bronchiseptica, and B.parapertussis, and B.pertussis specific assays that target the region among the most well characterized. ptxS1 Number target of single copy found in B. bronchiseptica, B. pertussis, and B. parapertussis IS1002 Number target of low copy found in B.pertussis and B.parapertussis hIS1001 Number target of low copy found in B.holmesii RecA Number target of single copy that is dependent on the probe design or primer design of B.bronchiseptica specific, B.pertussis, and B.parapertussis or B.holmesii specific. Various combination of the amplification of the amplification target in sequential or multiplex PCR can be of help in identifying the species of Bordetella. However, the maximum performance can only be possible if the used technology is designed to deal with various targets because there is sensitivity difference between the number target of high copy and number target of a single copy. For instance, the multiples IS481or IS1001 PCR can be tracked by the ptxA-Pr specific PCR when the outcome is positive and thus various outcomes can be gotten IS 1001 positive B.bronchiseptica or B.parapertussis IS481 positive ptxAprtnegative: Bordetella species IS481 positive ptxAPr positive: B.pertussis IS481positve & IS 1001 positive Possible coinfection with various Bordetella species. For purposes of practicality, a +ve IS481 PCR is preferred as B.pertussis infection that is probable, when the clinical symbols are according to the results. Additionally, a +ve IS1001 PCR outcome is considered to be the infection of the probable B.parapertussis. The epidemiological research cases with clinical data that are known, the positive outcomes from the IS481 PCR should only be taken as the infection evidence with the Bordetella spp. Some of the amplification targets employed include the IS481, IS1001, PTpromoter, ptxA-Pr, RecA, BP3385, IS1002, ptxS1, hIS1001, PtxP, and ptxA-Pr (Park, 2011).  The real time PCR details are outlined in various targets with each PCR running separately; IS 481 IS 1001, and ptxA-Pr. The IS 481 are number targets with high copy found I Some B, B.Pertussis, B. holmessi, and B.bronchiseptica. The IS 1001 are number targets High copy found in B. bronchiseptica, and B. parapertussis. The ptxA-Pr are number targets with a single copy found in B. bronchoseptica, B.pertussis, and B.parapertussis, but the assay will only be assigned specifically for the B. pertussis. The sample materials that will be used will comprise of the aspirated from the clients that have infection with B.parapertussis, B.pertussis. The apparatus used here are the pipettes, Micro centrifuge, and aerosol pipettes. Assessment Various quality control and quality assurance when employing the methods of PCR, among the assessment guidance are the UK Health Protection Agency and the United States Environmental Protection Agency. When coming up with the method of in-house PCR on the ABI benchmark, a perfect beginning point for this involves their respective guidance. The assessment of the LightCycler capillary system uses the World Health Organization manual. To prevent the crossover contamination, all the formats of the all real time PCR reduces the crossover contamination. When using the system of Uracil DNA glycosylate, additional lower level crossover contamination reduction is possible, and the generic reactions that are commercially available mixtures containing the fundamental reagents like the LightCyclerFastStart probes of DNA master hybridization (He, 1994). The PCR assays for the genes of human like mitochondrial cytochrome oxidase gene can be used to control the extracted samples. The internal process control needs to be included in every sample to assess the PCR inhibition. These can either be competitive or exogenous for instance phocine herpes virus. For the formats of real time PCR, the internal amplification control is preferred to avoid reporting that are false negative. The internal control according to the separate amplification of the herpes virus or on alternative mechanism to the PCR of PT promoter needs description. Almost every tested sample harbored by the sequences of human gene, and therefore, the amplification can be employed in inhibiting control (Ehrlich & Greenberg, 1994). It is important to test the inhibition presence to the reaction of PCR to avoid the reporting that are false negative. Assuming the IPC is not present, another alternative is to spearhead the replicate sample having the positive control DNA. For every test sample, a duplicate is prepared, the spiked samples has low dilution of the +ve control DNA on the already extracted sample. The two reactions are run and analyzed concurrently. Assuming the test sample do not contain the Bordetella detectable levels, and spiked reaction generates a signal that is comparable to those obtained from similar dilution employed in standard curve, then the outcome is reported as –ve result that is valid (Massay, 2005). When compared to similar dilution in standard curve, the spiked sample produces no signal then the outcome is reported as inhibitory. The in-run control entails the negative controls and the positive controls. Normally, the non-standardizes purified DNA or the bacterial suspension are the positive control. To ensure that the performance is continuous, the participation in the external quality assessment evaluation programme needs to be encouraged. In summary, the project involved using a real time PCR for diagnosis in the laboratory of human infection with a whooping cough. Bordetella pertussis causes a whooping cough; a bacterial infection that infects the respiratory. The disease is normally mild. Pertussis has remained to be an endemic in the entire world and is considered to A clear understanding of the biological functions of the recently be a disease that is cyclic, peaking after 3 to 5 years. One of the reasons behind the project was to come up with a document on the guidance that related to using real time PCR on the extracted DNA from the specimens found in clinics gotten from the clients that have whooping cough. It is proper to sample PCR justification by collecting nasopharyngeal aspirate or nasopharyngeal swab (Cheung, 2006). Either anterior nasal swabs or throat swabs should not be recommended because the bacteria of B.pertussis are mainly attached to ciliated epithelium found in the nasopharynx. Various commercial kits are found for the extraction of DNA. To be specific, the Qiagen (QIAamp DNA mini kit) and highly pure PCR template Roche are normally used. The protocol and the guidance are intended for using the real time PCR on DNA that was gotten from the clinical samples obtained from the patients who are suspected of having whooping cough or those infected with the nasopharyngeal and sampled from posterior nasopharynx. The Real-time PCR tools enable simultaneous multiple target detections since they have changing excitations of 6 or more varied detection of colors. Various apparatus are present for the real time PCR (McDonough et.al, 2005). Reference 1. Cheung, K. 2006. Development of a multiplex PCR assay for detection and differentiation of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica in Nasopharyngeal specimens. Hong Kong: [Dept. of Health Technology and Informatics, The Hong Kong Polytechnic University]. 2. Detecting PCR pathogens in real time. 2005. Idaho Falls, Idaho: Idaho National Laboratory. 3. Dorak, M. T. 2006. Real-time PCR. New York: Taylor & Francis. 4. Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications. 2010. NewYork: New York. 5. Edwards, K. 2004. Real-time PCR: an essential guide. Wymondham, Norfolk: Horizon Bioscience. 6. Ehrlich, G. D., & Greenberg, S. J. 1994.PCR-based diagnostics in infectious disease. Boston: Blackwell Scientific Publications. 7. He, Q. 1994. Diagnosis of Bordetella pertussis infection and investigation of pertussis immunity by PCR and EIA serology. Turku: Turun yliopisto. 8. Lázaro, D. 2013. Real-time PCR in food science: current technology and applications. Norfolk, UK: Caister Academic Press. 9. Logan, J., Edwards, K., & Saunders, N. 2009. Real-time PCR: current technology and applications. Norfolk, UK: Caister Academic Press. 10. Mackay, I. M. 2007. Real-time PCR in microbiology: from diagnosis to characterization. Norfolk, UK: Caister Academic. 11. Massay, S. C. 2005. A molecular diagnostic technique for bordetella pertussis using real-time PCR: implications for diagnosis and public health intervention in Alaska. New York: New York. 12. McDonough, E. A., Barrozo, C. P., Russell, K. L., & Metzgar, D. 2005. A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical Specimens. Ft. Belvoir: Defense Technical Information Center. 13. McPherson, M. J., & Møller, S. G. 2006.PCR 2nd ed.. New York: Taylor & Francis. 14. Park, D. J. 2011. PCR protocols 3rd ed.. New York, N.Y.: Humana Press. 15. Pestana, E. A. 2010. Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications. Dordrecht: Springer. Read More