At the Nunc A/S research laboratory an extensive testing of several systems for DIAPOPS (
) has been made. These systems are briefly described in the following text. 1. Introduction
At the Nunc A/S research laboratory an extensive testing of several systems for DIAPOPS ( ) has been made. These systems are briefly described in the following text.
Two systems have been tested for detection of Salmonella.
) and the DIAPOPS ( ) was made in collaboration with Dr. Jeffrey Hoorfar, Danish Veterinary Laboratory ( ). The DIAPOPS system recognized the purified template DNA in the first test, and no special optimization was necessary. The primer chosen as the solid phase primer was tested with both 0 T's and 10 T's as a linker in the 5'-end ( ). The solid phase primer with 10 T's improved the results when compared to the solid phase primer without any linker ( ). The system has been tested on pure cultures of Salmonella in the laboratory of Dr. Jeffrey Hoorfar. The results are as expected, and the DIAPOPS system recognize the tested Salmonella strains. ). The system detected the template plasmid in the first experiment using NucleoLink and DIAPOPS. No further optimization was necessary. The DNA used in the test was purified. The system is at present being tested in a quantitative PCR with an internal standard developed by Dr. Kim Holmstrøm, Biotechnological Institute, Denmark ( ). 3. PVY
A PCR system was developed for detection of Potato Virus Y (PVY) and optimized for DIAPOPS by Dr. Karen Husted, Danish Institute of Plant and Soil Science. The PCR was made directly from cDNA made from RNA extracted from virus particles. The particles were concentrated directly from raw potato material. In the first tests of the system with DIAPOPS there were problems with the reproducibility. In these tests there was no poly T-linker in the 5'-end, and the PCR was made in NucleoLink Strips not blocked with BSA prior to PCR. When a linker was added ( ) and BSA was used to block the wells ( ), the DIAPOPS detection performed well both in the Nunc A/S research laboratory and in the laboratory of Dr. Karen Husted.
The test has been evaluated in a small blind test with 10 real samples in cooperation with Dr. Steen Lykke Nielsen, Danish Institute of Plant and Soil Science ( ). The DIAPOPS system detected all 8 positive samples and none false positive. It is now under further evaluation and testing in the laboratory of Dr. Steen Lykke Nielsen.
4. HPV
The PCR primers for the detection of human papiloma virus (HPV) has been developed by the research group headed by Dr. Jan Walboomers at the Free University Amsterdam, The Netherlands (Jacobs et al., 1995). The system uses two HPV general primers for amplification, and distinguishes between low-risk and high-risk HPV using oligonucleotide probes. Due to the necessity of using the probe detection to characterize the amplified product, the DIAPOPS system is very useful in this system. The system has been tested in DIAPOPS, and the DIAPOPS detection was successful after a decrease in incubation time in the PCR program from approximately one minute to 20 seconds ( ) and blocking the wells with BSA before PCR ( ). The system is now under evaluation in the laboratory of Dr. Jan Walboomers ( ).
5. BLV
An assay for detection of bovine leukemia virus (BLV) DNA with DIAPOPS has previously been described {Rasmussen et al., 1991}. The assay is very stable, functions well, and is the Nunc in-house standard. It is also used in the quality control test of all new productions of NucleoLink ( ). The assay has not been used with NucleoLink Strips on real samples, only with the target sequence cloned into a plasmid.
6. Phytophtora fragariae
A PCR system developed by Dr. Peter Bonants, Research Institute for Plant Protection, The Netherlands ( ), for detection of the fungi Phytophtora fragariae. The primers recognize a ribosomal DNA sequence on the genome. The analysis using DIAPOPS did not perform well in the first tests, but the results were improved after addition of a poly T-linker to the solid phase primer ( ), a decrease in the annealing temperature from 72ºC to 65ºC ( ), and blocking with BSA of the coated NucleoLink Strips prior to PCR ( ). The system is now under evaluation at the laboratory of Dr. Peter Bonants.
In the development phase of this systems several problems were encountered. The solid phase primer gave rise to a very high background ( ), and the hybridization signal was lower in the first hybridization than in the following hybridizations ( ). The problem with the high background was solved by purchasing a new solid phase primer of the same sequence. The rehybridization problem probably arose because the target sequence was from a ribosomal gene. The single stranded PCR product was actually designed to form secondary structures, and probably also does this when attached to a solid phase. For this reason hybridization is difficult with special PCR products coming from sequences likely to make secondary structures.
7. cDNA from mRNA coding for rat brain actin
The primers in this system recognize cDNA synthesized from mRNA from rat brain actin. The system was developed by Dr. Brands from Solvay Duphar. B.V., The Netherlands ( ). The system worked in DIAPOPS in the first test, and is at present under evaluation in Dr. Brands' laboratory.
8. Lambda-DNA
In order to incorporate a new quality control system for NucleoLink Strips with a well defined target DNA, a PCR for the detection of purified DNA from the Lambda-bacteriophage was developed at the research laboratory of Nunc A/S. The liquid phase DNA amplification worked in the first experiment, but the DIAPOPS analysis was negative. An analysis by direct hybridization to the solid phase primer by a labelled complimentary probe ( ) showed that no solid phase primer was bound to the surface. The problem was solved by purchasing a new solid phase primer with the same sequence. This indicates that the problem may have been that the solid phase primer was not 5'-end phosphorylated. In the first experiment with the new solid phase primer, the DIAPOPS analysis performed well, and the system is now under careful testing to ensure its stability when used in the quality control.
9. cDNA from rat IL2 receptor mRNA
The primers recognize cDNA formed from interleukin 2 receptor alpha chain mRNA extracted from rat lymph nodes. The system was tested by Liselott Svendson at the University of Göteborg ( ). In the first tests, there was a very high background, and positive DIAPOPS signals could only be seen in a very few experiments ( ). The background problem was traced to an unspecific binding of the biotin recognizing compound used (extravidine-alkaline phosphatase conjugate) ( ). When testing another synthesis of the same primer sequence, and using a streptavidin-AP conjugate from another source, the problem was solved, and the system performed as expected in the DIAPOPS ( ). The system is currently used in the laboratory of Esbjørn Telemo at the University of Göteborg.
10. Conclusion
All nine systems tested proved to work in the DIAPOPS assay. This shows it is very likely that all standard PCR systems now used with only detection by agarose gel or detection by other means of hybridization, such as Southern blotting, can be transferred to DIAPOPS. The DNA amplification step and the hybridization step in the DIAPOPS procedure differs little from the standard PCR and hybridization detection used, but will make the workload of the analysis smaller, especially when many samples are to be analyzed.
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