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PCR

PCR

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PCR

Introduction

The following study is a research and analysis test on three different samples of cerebrospinal fluid of different individuals. Previous tests raised the possibility of herpes simplex virus (HSV) related encephalitis. The test activity will involve DNA extraction from the cerebrospinal fluid and run a test for herpes simplex virus by polymerase chain reaction (PCR). The study will also involve investigating the better of three different PCR approaches to detect HSV in the sample DNA samples. The main objective of this study is to detect the presence of herpes simplex virus in the three samples as a means of establishing the cause of encephalitis (Mullis, 2004). Additionally, this study will involve valid and informed research on three different PCR approaches and establish the most appropriate for routine diagnostic work.

Materials and Methods

The materials in this exercise include three cerebrospinal fluid samples from three different patients; GU01, GO02, and GU03. Three HSV testing kits including real time PCR using hybridization probes on the LightCycler, real time PCR using SYBR green on the Rotorgene, and time PCR using Taqman probes on Rotorgene.

DNA Extraction

A molecular assay for the homebrew assay and a DNA kit requiring 200 ?l and is to be used in accordance with the instructions set by the manufacturer. For assay on real time PCR, there will be need for rapid DNA extraction protocol. A 100 ?l of sample will be added to a 1.5 ml tube and then to a 300 ?l of a suspension. This suspension will include twenty percent Chelex 100 resin, in 0.1 mM EDTA–0.1% sodium azide and 10 mM Tris-HCl (pH 8.0). After vortexing the tube sample for ten seconds, ten-minute incubation at 100 degrees should follow. The tube will then be allowed to cool to cool to room temperature. After the complete settling of the resin, there will be amplification of 5 ?l of the supernatant.

Real time PCR Using Hybridisation Probes on the LightCycler

The method involves the use of two labeled oligonucleotides. The probes in this kit are simple to use and design. They prove to be effective when applied to real time quantification and are sensitive to cell mutation through the implementation of melting curves with high resolution. Rapid fluorescence and cycling allows complete analysis and amplification in a period spanning less than half an hour.

Real time PCR Using Taqman Probes on Rotorgene

This kit is fitted with a Rotor-Gene Probe PCR Kit made to incorporate the Rotor Gene Q as well as other Rotor Gene Cyclers. This facilitates sensitive cDNA target quantification through the use RT-PCR using probes on a specified sequence (Carter, 2010). Optimum performance is achieved when a combination between the Rotor Gene cylinder and a master mix of specialized design is used. The master mix should be stored at a range of two to eight degrees to ensure convenience (Carter, 2010).

Real Time PCR Using SYBR Green on the Rotorgene

This test kit provides real time and rapid genomic quantification of genomic RNA, cDNA, and DNA targets on the Q rotor gene in a convenient and simple handling format. However, this kit is incompatible with rotor genes especially Rotor Gene 6000 and Rotor Gene 3000. The SYBR Green I dye in the master mix has the ability of analyzing different targets without necessarily synthesizing probes with specified tags (Feng, 2001). High sensitivity and specificity in PCR is achieved through the implementation of enzyme hot start.

Results

For all the three samples, they exhibited a pass for the extraction/ inhibition control PCR. In the SYBR green real time PCR, both samples GU01 and GU02 positive sloping melting curves apart from sample GO03 that exhibited a negative sloping melting curve. For the duplex TaqMan real time PCR, samples GU01 and GU03 exhibited negative results on the HSV-2 yellow channel reaction with sample GU02 exhibiting a positive result. Samples GU02 and GU03 posted negative HSV-1 results on the green channel reaction apart from sample GU01 that posted a posted a positive result on the same. For the HSV hybridization probe real time PCR tests, samples GU01 and GU02 were both positive HSV type one virus. Sample GU03 exhibited negative results however.

PCR results for samples: HSV hybridization probe real-time PCRHSV duplex TaqMan real-time PCRHSV SYBR-green real-time PCRExtraction / inhibition control PCRSampleAmplification curve

(POS or NEG)

Melting curve

(HSV type 1 or 2)

HSV-1 reaction

(green channel)

(POS or NEG)

HSV-2 reaction

(yellow channel)

(POS or NEG)

Amplification curve

(YES or NO)

Melting curve

(POS or NEG)

(PASS or FAIL)

GU01PositiveType 1PositiveNegativeYesPositivePassGU02PositiveType 2NegativePositiveYesPositivePassGU03NegativeType 1NegativeNegativeYesNegativePass Additional Quality Control results HSV hybridization probe real-time PCRHSV duplex TaqMan real-time PCRHSV SYBR-green real-time PCRControlAmplification curve

(PASS or FAIL)

Melting curve

(PASS or FAIL)

HSV-1 reaction

(green channel)

(PASS or FAIL)

HSV-2 reaction

(yellow channel)

(PASS or FAIL)

Amplification curve

(PASS or FAIL)

Melting curve

(PASS or FAIL)

NegPassPassPassPassFailPassNegPassPassPassPassFailPassNegPassPassPassPassFailPassHSV 1

POS

PassPassPassPassPassPassHSV 2

POS

PassPassPassPassPassPass

Discussion and Conclusion

The polymerase chain reaction is a powerful tool with excellent sensitivity that allows DNA molecule detection. PCR sensitivity is normally complemented by specified hybridization techniques. For diagnosing HSV virus with real time PCR using hybridization probes on the LightCycler, this method relies on five to three LightCycler Polymerase activities when cleaving dual labeled probes in the course of hybridization to the fluorophore based detection and complementary target sequence (Feng, 2001). Similar to the other methods in PCR, the resulting signal from the fluorescence allows accumulated quantitative measurements during the PCR exponential stages. However, in this case, the LightCycler brings about a significant advantage of increasing the detection specificity.

Real Time PCR Using SYBR Green on the Rotorgene, this method offers real time and rapid genomic quantification of genomic RNA, cDNA, and DNA targets on the Q rotor gene in a convenient and simple handling format. The SYBR Green I dye in the master mix has the ability of analyzing different targets without necessarily synthesizing probes with specified tags. High sensitivity and specificity in PCR is achieved through the implementation of enzyme hot start.

Another advantage of this method is that these kits are fitted with PCR cyclers with Rotor Gene real time. This concept employs an exceptional design of centrifugal rotation. However, this method requires the simultaneous application of several target sequences in one given reaction (Khodari, 2005).

The other possible method HSV diagnosis is the real time PCR using TaqMan probes on Rotorgene. This method of diagnosing HSV facilitates sensitive cDNA target quantification through the use RT-PCR using probes on a specified sequence. Optimum performance is achieved when a combination between the Rotor Gene cylinder and a master mix of specialized design is used. The master mix should be stored at a range of two to eight degrees to ensure convenience (Khodari, 2005). Additionally, TaqMan utilizes both a system and concept fair to understand and use. Additionally, the performing PCR using TaqMan probes demonstrates sufficient sensitivity, monitoring in quantitative form, and can be used as a tool in examining microbial and periodontal disease. The other advantage of this kit is that it can be detect mutations of microorganisms in the test samples and would advice on which prompt action to take. The only limitations hindering this method are its expensive cost and expertise needed to learn the complicated procedures of applying it.

In this case, PCR tubes are fixed onto rotor blades that spin the tubes past the same detector and light source excitation in moving air chamber. This implies that minimal temperature and minimal optical variation occurs between the tubes. Despite the above benefits, this method has its limitations. This method is only limited to one use and cannot be implemented for prevention, diagnosis, or disease treatment.

With the above consideration, I was able to establish that the real time PCR using TaqMan probes on Rotorgene was the most convenient method to implement in the lab because it has multiple uses as well as minimal limitations compared to the other methods.

References

Bartlett, J. M. S., & Stirling, D. (2003). PCR protocols. Totowa, N.J: Humana Press.

Carter, I. W. J., Schuller, M., James, G. S., Sloots, T. P., & Ebooks Corporation. (2010). PCR for Clinical Microbiology. Dordrecht: Springer.

Dieffenbach, C. W., & Dveksler, G. S. (2005). PCR primer: A laboratory manual. Plainview, N.Y: Cold Spring Harbor Laboratory Press.

Feng, P., & Read, G. S. (2001). MRNA decay during HSV infections: Interactions between a viral nuclease and cellular translation factors.

Innis, M. A. (2000). PCR protocols: A guide to methods and applications. San Diego: Academic Press.

Khodari, Y. A. M., & University of Manchester. (2005). Quantification of Herpes simplex virus type 1(HSV-1) DNA by the polymerase chain reaction. Manchester: University of Manchester.

Mullis, K. B., Ferre?, F., & Gibbs, R. (2004). The Polymerase chain reaction. Boston: Birkha?user.

Polymerase chain reaction. (2005). Noisy-le-Grand: C.M.B. Association-Paris.

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