Validationreport Clavibacter michiganensis michiganensis Ptssk primers and probe

Transcription

1 Validationreport Clavibacter michiganensis michiganensis Ptssk primers and probe This report is the summary of a validation research of the new primers for the plant pathogenic bacteria Clavibacter michiganensis michiganensis (Cmm) developed by Rijk Zwaan Marjolein Spiekerman Sven Berendsen Jessica Oosterhof Rijk Zwaan

2 Methods General information Development of the primers and validation started in February of The primers target the putative two-component system sensor kinase (Ptssk) gene which is located at the to basepair of Clavibacter michiganensis subsp. michiganensis NCPPB 382 complete genome. Blast searches showed this gene to be specific for Cmm. A Minor Groove binding probe was used for specificity. Total amplicon size is 132 basepairs. Millipore Milli-Q Biocel was used in the PCR reaction mix. Primers were developed by S. Berendsen at Rijk Zwaan (poster in press for the APS meeting in August 2011: The development of a specific Real-Time TaqMan for the detection of Clavibacter michiganensis supsp. michiganensis ) Sample preparation DNA of the Cmm collection was extracted by Naktuinbouw with the Dneasy Tissue Kit from Qiagen. This purified DNA of both true pathogenic Cmm species and Cmm lookalikes was used to test the specificity of the developed primers. PCR development This validation report is to test the specificity and sensitivity for the following primers and probe based on the putative two-component system sensor kinase gene. Forward primer RZ_ptssk 10 5'-GGGGCCGAAGGTGCTGGTG-3' Reverse Primers RZ_ptssk 11 5'-CGTCGCCCGCCCGCTG-3' Probe RZ_ptssk 12 6FAM-TGGTCGTCCTCGGCG-MGBNFQ The PCR program shown in Table 2 was used on a C1000 Thermal Cycler CFX96 Real Time System from Biorad for the Real Time PCR. Table 1. PCR reaction mix PCR-volumia (µl) 1x RZ_ptssk 10 (20 pmol/µl) 0,3 RZ_ptssk 11 (20 pmol/µl) 0,3 RZ_ptssk 12 (20 pmol/µl) 0,3 TaqMan master mix (2x) Roche 12,5 mq 10,60 DNA 1,0 Total volume 25 Table 2. PCR program Step Temperature ( C) Duration (min) Repeat x from step 2

3 Results Table 3 Overview of specificity of the Ptssk primers on DNA of the Naktuinbouw Cmm collection Nakt isolate Nakt isolate number Bioassay* PTSSK-12 MGB Ta=60 number Bioassay* PTSSK-12 MGB Ta=60 1 C 20,12 36 negative N/A 2 C en + 18,78 37 negative N/A ,76 38 negative N/A 4 C 20,48 39 negative N/A 5 C 20,25 40 negative N/A 6 C 19,79 41 negative N/A 7 C en + 18,11 42 negative N/A ,81 43 negative N/A 9 C 20,15 44 negative N/A ,11 45 negative N/A 11 C 21,86 46 negative N/A ,32 47 negative 20,28 13 C en + 21,40 48 negative N/A ,46 49 negative 39, ,64 50 negative N/A 16 + N/A 51 negative N/A ,10 52 negative N/A 18 C 20,29 53 negative 36, ,57 54 negative 37,46 20 C 20,39 55 negative N/A 21 negative N/A 56 negative N/A 22 negative 38,16 57 negative N/A 23 negative N/A 58 C en + 19,29 24 negative 36,51 59 C 23,98 25 negative 35,77 60 negative 27,37 26 C en + 22,27 61 negative N/A 27 negative N/A 62 C en + 20,13 28 negative N/A 63 negative N/A 29 negative N/A 64 negative 38,37 30 negative N/A 65 PD 22,03 31 negative N/A 66 PD 21,11 32 negative 38,84 67 PD 21,37 33 negative N/A 34 negative N/A 35 negative N/A

4 Specificity Naktuinbouw made a collection of the different Cmm isolates obtained from their own lab and seed companies in The Netherlands. The isolates were tested in a bioassay to see if the isolate was pathogenic. DNA from these isolates was purified and used for the development of primers. Table 3 shows an overview of the isolates, their pathogenicity in the bioassay and the reaction in the Ptssk Real Time PCR. In addition the Rijk Zwaan Cmm collection (7 positive Cmm s and 17 lookalikes) was tested with the ptssk primers, which all gave correlating results in the PCR assay. Also other ISHI participants tested the primers in the last 2 years, and only positive feedback was given. The overall impression is that the PCR correlates very well to the pathogenicity. However there are a few exceptions in the Naktuinbouw Cmm collection. Isolate 16 for example, is negative in PCR and positive in bioassay. In the file provided by the Naktuinbouw it can be seen that many primers react negative to this isolate. Therefore it would be good to look into this specific isolate and test it more thoroughly, by AFLP for example. This in order to confirm if we are dealing with a true pathogen and investigate how closely related it is to other Cmm isolates. Another difference is isolate 47, positive in PCR and negative in bioassay. The Naktuinbouw file shows that all Cmm primers test positive for this isolate. Sensitivity A dilution series of 3 Cmm isolates was made in 0.05 M Phosphate buffer. For this experiment 3 different true Cmm isolates were selected, 2 of them are reference isolates from the Naktuinbouw, the last one is the isolate Rijk Zwaan spikes the seed extract with, before plating on selective medium in the Cmm protocol. Each sample was diluted 8 times in a 10-fold dilution. From each dilution was plated on the non selective medium YDC before the samples were incubated for 5 minutes at 100 C to release DNA. In the PCR essay was used per dilution. Table 4 shows the result of the sensitivity of the PCR and plate assay of 3 dilution series. When considering the 100x volume increase in the plate assay it is clear that the sensitivity of the PCR is almost comparable to the Real Time PCR. Table 4. Sensitivity plate versus RealTime PCR Dilution Cmm 500 Naktuinbouw reference isolate Ct value #colonies PCR on YDC Cmm 501 Naktuinbouw reference isolate Ct value PCR #colonies on YDC Cmm ZUM 3059 Ct value PCR 0 x x x x x x N/A 320 N/A 140 N/A x N/A 29 N/A 19 N/A x N/A N/A N/A #colonies on YDC Repeatability and Reproducibility In order to test the reproducibility of the experiment 8 isolates of the Naktuibouw Cmm DNA collection were selected to test in PCR. Of the 8 selected isolates 4 were Cmm lookalikes and 4 were true pathogenic Cmm isolates. The isolates were tested in duplo on the same PCR plate and, at another time, the isolates were tested again to determine reproducibility. The Ct result of these PCR reactions are summarized in Table 5.

5 Table 5. RealTime PCR repeatability and reproducability PCR A PCR B PCR C Nakt isolate Data from validation file of Nakt Repeat Duplo of the repeat Reproducability number: PTSSK-12 MGB Ta=60 Ct Ct Ct 1 Cmm 20,12 21,67 21,72 21,51 2 Cmm 18,78 20,40 20,31 20,18 3 Cmm 18,76 20,93 20,80 20,74 4 Cmm 20,48 22,78 22,63 22,63 27 Lookalike N/A N/A N/A N/A 28 Lookalike N/A N/A N/A N/A 29 Lookalike N/A N/A N/A N/A 30 Lookalike N/A N/A N/A N/A Conclusion This report shows that the Ptssk primers meet all validation criteria. They are specific, sensitive, the repeatability as well as the reproducibility are good. There are 2 isolates that give an odd result when comparing the Real Time PCR to the bioassay, it is desirable to identify these isolates better. These differences however, occur with more primers pairs and therefore do not reduce the value of these ptssk primers, which are more specific primers in comparison to the ones used now.