Cladosporium cladosporioides PCR Detection Kit Product # 33000

Transcription

1 3430 Schmn Parkway Thrld, ON, Canada L2V 4Y6 Phne: (905) Fax: (905) Cladsprium cladspriides PCR Detectin Kit Prduct # Prduct Insert Pathgen Infrmatin Cladsprium is ne f the mst widespread mlds. It includes abut 40 species naturally fund in sil, n decaying plant material and as plant pathgens. Cladsprium rt (Cladsprium spp.) f grapevine (Vitis vinifera) is a cmmn disease, particularly in Cabernet Sauvignn and ther red wine grape cultivars. It is favred by delayed harvest t btain the phenlic maturity necessary fr highquality red wine. Symptms appear n mature grapes and are characterized by berry dehydratin, a firm decay affecting a small prtin f the berry and a superficial live-green mld. Rapid and accurate detectin f Cladsprium infectins is highly imprtant t facilitate the mnitring f Cladsprium in plant samples. Principle f the Test Nrgen s Cladsprium cladspriides PCR Detectin Kit cnstituents a ready-t-use system fr the islatin and detectin f C. cladspriides using end-pint PCR. The kit first allws fr the islatin f fungal DNA frm the plant samples using spin-clumn chrmatgraphy based n Nrgen s prprietary resin. Fungal DNA can be islated frm fungi grwing n culture plates, r frm plant tissue r fruit using this kit. The DNA is islated free frm inhibitrs, and can then be used as the template in a PCR reactin fr C. cladspriides detectin using the prvided C. cladspriides Master Mix. The C. cladspriides Mastermix cntains reagents and enzymes fr the specific amplificatin f a 320 bp regin f the fungal genme. In additin, Nrgen s C. cladspriides PCR Detectin Kit cntains a secnd Mastermix, the PCR Cntrl Master Mix, which can be used t identify pssible PCR inhibitin and/r inadequate islatin via a separate PCR reactin with the use f the prvided PCR cntrl (PCRC) r Islatin Cntrl (IsC), respectively. This kit is designed t allw fr the testing f 24 samples. Kit Cmpnents: Cmpnent Lysis Slutin Wash Slutin Elutin Buffer Cntents 15 ml 9 ml 3 ml Bead Tubes 24 Spin Clumns 24 Cllectin Tubes 24 Elutin tubes (1.7 ml) 24 C. cladspriides 2x PCR Master Mix 0.35 ml Cntrl 2x PCR Master Mix Islatin Cntrl (IsC) *a 0.35 ml 0.3 ml C. cladspriides Psitive Cntrl (PsC) *b 0.1 ml Nuclease Free-Water Nrgen s DNA Marker 1.25 ml 0.1 ml Prduct Insert 1 * IsC = Islatin Cntrl ; PsC= Psitive Cntrl a The islatin cntrl is a clned PCR prduct. b The psitive cntrl is C. cladspriides genmic DNA

2 Custmer-Supplied Reagents and Equipment Dispsable pwder-free glves Benchtp micrcentrifuge 1.5 ml micrcentrifuge tubes 65 C water bath r heating blck % ethanl 70% ethanl Lyticase (ptinal) Strage Cnditins and Prduct Stability All buffers shuld be kept tightly sealed and stred at rm temperature (15-25 C). Buffers can be stred fr up t 1 year withut shwing any reductin in perfrmance. The C. cladspriides 2x PCR Master Mix, Cntrl 2x PCR Master Mix, C. cladspriides Psitive Cntrl (PsC) and the Islatin Cntrl (IsC) shuld be kept tightly sealed and stred at -20 C fr up t 1 year withut shwing any reductin in perfrmance. Repeated thawing and freezing (> 2 x) shuld be avided, as this may reduce the sensitivity. If the reagents are t be used nly intermittently, they shuld be frzen in aliquts. General Precautins The user shuld exercise the fllwing precautins when using the kit: Use sterile pipette tips with filters. Stre and extract psitive material (specimens, cntrls and amplicns) separately frm all ther reagents and add it t the reactin mix in a spatially separated facility. Thaw all cmpnents thrughly at rm temperature befre starting an assay. When thawed, mix the cmpnents and centrifuge briefly. Wrk quickly n ice. Quality Cntrl In accrdance with Nrgen s ISO 9001 and ISO certified Quality Management System, each lt f Nrgen s C. cladspriides 2x PCR Master Mix, Cntrl 2x PCR Master Mix, C. cladspriides Psitive Cntrl (PsC) and the Islatin Cntrl (IsC)are tested against predetermined specificatins t ensure cnsistent prduct quality. Prduct Use Limitatins Nrgen s C. cladspriides PCR Detectin Kit is designed fr research purpses nly. It is nt intended fr human r diagnstic use. Prduct Warranty and Satisfactin Guarantee NORGEN BIOTEK CORPORATION guarantees the perfrmance f all prducts in the manner described in ur prduct manual. The custmer must determine the suitability f the prduct fr its particular use. Safety Infrmatin Ensure that a suitable lab cat, dispsable glves and prtective gggles are wrn when wrking with chemicals. Fr mre infrmatin, please cnsult the apprpriate Material Safety Data Sheets (MSDSs). These are available as cnvenient PDF files nline at CAUTION: DO NOT add bleach r acidic slutins directly t the sample-preparatin waste.

3 Prtcl A. Cladsprium cladspriides Genmic DNA Islatin Imprtant Ntes Prir t Beginning Prtcl: A variable speed centrifuge shuld be used fr maximum kit perfrmance. If a variable speed centrifuge is nt available a fixed speed centrifuge can be used, hwever reduced yields may be bserved. Ensure that all slutins are at rm temperature prir t use, and that n precipitates have frmed. If necessary, warm the slutins and mix well until the slutins becme clear again. Prepare a wrking cncentratin f the Wash Slutin by adding 21 ml f % ethanl (prvided by the user) t the supplied bttle cntaining the cncentrated Wash Slutin. This will give a final vlume f 30 ml. The label n the bttle has a bx that may be checked t indicate that the ethanl has been added. Lysate can be prepared frm either fungi grwing n plates, plant tissue r fruit. Please ensure that yu fllw the prper prcedure fr lysate preparatin in Step 1a. Fr the islatin f genmic DNA frm fungi grwing n plates, Cllectin Slutin must be prepared. Cllectin Slutin cnsists f 0.9% (w/v) NaCl prepared with distilled water. Preheat a water bath r heating blck t 65 C. Islatin Cntrl (IsC) An Islatin Cntrl (IsC) is supplied. This allws the user t cntrl the DNA islatin prcedure. Fr this assay, add the Islatin Cntrl (IsC) t the lysate during the islatin prcedure The Islatin Cntrl (IsC) must nt be added t the sample material directly. D nt freeze and thaw the Islatin Cntrl (IsC) mre than 2 times. The Islatin Cntrl (IsC) must be kept n ice at all times during the islatin prcedure. The PCR cmpnents f the C. cladspriides PCR Detectin Kit shuld remain at -20 C until DNA is extracted and ready fr PCR amplificatin. 1. Lysate Preparatin a. Fungi Grwing n Plates: Add apprximately 5 ml (vlume can be adjusted based n density f fungal grwth) f Cllectin Slutin (see ntes befre use) t the plate and gently cllect fungal spres and mycelium with an inculatin lp r autclaved pipette tip, ensuring nt t cllect any agar debris. Transfer up t 1 ml f washed spres and wet mycelium t a micrcentrifuge tube (prvided by user). Fungi frm Plant Tissue r Fruit: Wash the tissue r fruit with an apprpriate amunt f DNAse free water with vrtexing. Transfer up t 1 ml f washed spres and wet mycelium t a micrcentrifuge tube (prvided by user). b. Centrifuge at 14,000 x g (~14,000 RPM) fr 1 minute t pellet the cells. Pur ff the supernatant carefully s as nt t disturb r disldge the cell pellet. c. Add 500 L f Lysis Slutin t the cell pellet. Resuspend the cells by gentle vrtexing. d. Transfer the mixture t a prvided Bead Tube and secure the tube hrizntally n a flat-bed vrtex pad with tape, r in any cmmercially available bead beater equipment (e.g. Scientific Industries Disruptr Genie TM ). e. Vrtex fr 5 minutes at maximum speed r ptimize the cnditin fr any cmmercially available bead beater equipment. Nte: Faming during the hmgenizatin is cmmn. This faming is due t detergents present in the Lysis Buffer and will nt affect the prtcl. f. Incubate the Bead Tube with lysate at 65 C fr 10 minutes. Occasinally mix the lysate 2 r 3 times during incubatin by inverting the tube.

4 g. Briefly spin the tube t remve liquid frm the cap, and transfer all f the lysate, including cell debris, t a DNase-free micrcentrifuge tube (prvided by the user) by pipetting. Ensure that the beads are nt transferred during the pipetting. h. Centrifuge the tube fr 2 minute at g (~14,000 RPM). i. Carefully transfer clean supernatant t a new DNase-free micrcentrifuge tube (prvided by the user) withut disturbing the pellet. Nte the vlume. j. Add an equal vlume f 70% ethanl (prvided by the user) t the lysate cllected abve (100 µl f ethanl is added t every 100 µl f lysate). Vrtex t mix. k. Prceed t Step 2: Binding t Clumn 2. Binding DNA t Clumn a. Assemble a spin clumn with ne f the prvided cllectin tubes. b. Add 10 L f Islatin Cntrl (IsC) t the lysate mixture. c. Apply up t 600 µl f the lysate with ethanl nt the clumn and centrifuge fr 1 minute at 14,000 x g (~14,000 RPM). Discard the flwthrugh and reassemble the spin clumn with the cllectin tube. Nte: Ensure the entire lysate vlume has passed thrugh int the cllectin tube by inspecting the clumn. If the entire lysate vlume has nt passed, spin fr an additinal minute. d. Depending n yur lysate vlume, repeat step 2C if necessary. 3. Clumn Wash a. Apply 500 µl f Wash Slutin t the clumn and centrifuge fr 1 minute. Nte: Ensure the entire wash slutin has passed thrugh int the cllectin tube by inspecting the clumn. If the entire wash vlume has nt passed, spin fr an additinal minute. b. Discard the flwthrugh and reassemble the clumn with its cllectin tube. c. Repeat step 3a t wash clumn a secnd time. d. Discard the flwthrugh and reassemble the spin clumn with its cllectin tube. e. Spin the clumn fr 2 minutes in rder t thrughly dry the resin. Discard the cllectin tube. 4. DNA Elutin a. Place the clumn int a fresh 1.7 ml Elutin tube prvided with the kit. b. Add 75 µl f Elutin Buffer t the clumn. c. Centrifuge fr 2 minutes at 200 x g (~2,000 RPM), fllwed by a 1 minute spin at 14,000 x g (~14,000 RPM). Nte the vlume eluted frm the clumn. If the entire vlume has nt been eluted, spin the clumn at 14,000 x g (~14,000 RPM) fr 1 additinal minute. 5. Strage f DNA The purified DNA may be stred at 20 C fr a few days. It is recmmended that samples be placed at 70 C fr lng term strage.

5 B. C. cladspriides PCR Assay Preparatin Ntes: Befre use, suitable amunts f all PCR cmpnents shuld be cmpletely thawed at rm temperature, vrtexed and centrifuged briefly. The amunt f C. cladspriides 2X PCR Master Mix and Cntrl 2X PCR Master Mix prvided is enugh fr up t 32 PCR reactins (24 sample PCR, 4 psitive cntrl PCR and 4 n template cntrl PCR). Fr each sample, ne PCR reactin using the C. cladspriides 2X PCR Mastermix and ne PCR reactin using Cntrl 2X PCR Mastermix shuld be set up in rder t have a prper interpretatin f the results. Fr every PCR run, ne reactin cntaining C. cladspriides Psitive Cntrl and ne reactin as n template cntrl must be included fr prper interpretatin f results. The recmmended minimum number f DNA samples tested per PCR run is 6. Using a lwer vlume frm the sample than recmmended may affect the sensitivity f C. cladspriides Limit f Detectin. 1. Prepare the PCR fr sample detectin as shwn in Table 1 belw. The recmmended amunt f sample DNA t be used is 2.5 µl. Hwever, a vlume between 1 and 10 µl f sample DNA may be used as template. Adjust the final vlume f the PCR reactin t 20 µl using the Nuclease-Free Water prvided. Prepare the PCR reactin fr sample detectin (Set #1, using C. cladspriides 2X PCR Mastermix) and the PCR reactin fr cntrl detectin (Set #2, using Cntrl 2X PCR Mastermix) as shwn in Table 1 belw. The recmmended amunt f sample DNA t be used is 2.5 µl. Hwever, a vlume between 1 and 5 µl f sample DNA may be used as template. Ensure that ne C. cladspriides detectin reactin and ne cntrl reactin is prepared fr each DNA sample. Adjust the final vlume f the PCR reactin t 20 µl using the Nuclease-Free Water prvided. Table 1. PCR Assay Preparatin PCR Cmpnents C. cladspriides 2X PCR Master Mix Or Cntrl 2X PCR Master Mix Vlume Per PCR Reactin 10 µl Sample DNA 2.5 µl Nuclease-Free Water 7.5 µl Ttal Vlume 20 µl 2. Fr each PCR set, prepare ne psitive cntrl PCR as shwn in Table 2 belw: Table 2. PCR Psitive Cntrl Preparatin PCR Cmpnents C. cladspriides 2X PCR Master Mix Or Cntrl 2X PCR Master Mix Vlume Per PCR Reactin 10 µl C. cladspriides Psitive Cntrl (PsC) 10 µl Ttal Vlume 20 µl

6 3. Fr each PCR set, prepare ne n template cntrl PCR as shwn in Table 3 belw: Table 3. PCR Negative Cntrl Preparatin PCR Cmpnents C. cladspriides 2X PCR Master Mix Or Cntrl 2X PCR Master Mix Vlume Per PCR Reactin 10 µl Nuclease-Free Water 10 µl Ttal Vlume 20 µl C. C. cladspriides PCR Assay Prgramming 1. Prgram the thermcylcer accrding t the prgram shwn in Table 4 belw. 2. Run ne step PCR. Table 4. C. cladspriides Assay Prgram PCR Cycle Step Temperature Duratin Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 (40x) Step 2 60 C 15 sec Step 3 72 C 30 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C D. C. cladspriides PCR Assay Results Interpretatin 1. Fr the analysis f the PCR data, the entire µl PCR Reactin shuld be laded n a 1X TAE 1.7% Agarse DNA gel alng with 10 L f Nrgen s DNA Marker (prvided). 2. The PCR prducts shuld be reslved n the 1X TAE 1.5% Agarse gel at 150V fr 30 minutes (Gel running time will be vary depending n an electrphresis apparatus). 3. Sample results are prvided belw:

7 Figure 1: Detectin f C. cladspriides using the C. cladspriides PCR Detectin Kit. A representative 1X TAE 1.5% agarse gel shwing the amplificatin f serially diluted C. cladspriides psitive (lane 1 t 4) negative (lane NTC) cntrls. The size f the C. cladspriides target amplicn crrespnds t 320 bp as represented by the prvided DNA Marker (M). M NTC Islatin Cntrl PCR Cntrl Figure 2: A representative 1X TAE 1.5% agarse gel shwing the amplificatin f Islatin Cntrl and PCR Cntrl under different cnditins using the 2X PCR Cntrl Mastermix. The size f the Islatin Cntrl amplicn and PCR Cntrl amplicn crrespnd t 499 bp and 150 bp, respectively, as represented by the prvided DNA Marker (M). Lanes 1 t 5 shwed detectin f bth Islatin Cntrl and PCR Cntrl, suggesting that the DNA islatin as well as the PCR reactin was successful. Lane 6 shwed nly the detectin f PCR Cntrl suggesting that while the PCR was successful, the islatin failed t recver even the spiked-in Islatin cntrl. NTC = Negative Cntrl.

8 Table 5. Interpretatin f PCR Assay Results Input Type Target reactin Cntrl Reactin Interpretatin C. cladspriides Target Band (320 bp) IsC Band (499 bp) PCRC Band (150 bp) Psitive Cntrl X X X Valid Negative Cntrl X Valid Sample X X X Psitive Sample X X Negative Sample X Re-test Sample Re-test Sample X Negative Sample X X Psitive Sample X X Psitive Sample X Re-test ** Fr results btained that are nt cvered in Table 5 abve, please refer t the Trubleshting Sectin. E. C. cladspriides PCR Assay Specificity and Sensitivity The specificity f Nrgen s C. cladspriides PCR Detectin Kit is first and fremst ensured by the selectin f the C. cladspriides -specific primers, as well as the selectin f stringent reactin cnditins. The primers were checked fr pssible hmlgies t all in GenBank published sequences by sequence cmparisn analysis. The specific delectability f all relevant strains has thus been ensured by a database alignment and by PCR amplificatin with the fllwing bacteria cmmnly fund in filed samples. Aspergillus niger Cladsprium sp. Btrytis cinerea Mucr racemsus Alterneria tenuissima Rhizpus ryzae Penicillum sp. Fusarium xysprum

9 F. Linear Range The linear range f Nrgen s C. cladspriides PCR Detectin Kit was determined by analysing a dilutin series f a C. cladspriides quantificatin standards ranging frm 100 fg t 1 ng. Each dilutin has been tested in replicates (n = 4) using Nrgen s C. cladspriides PCR Detectin Kit n a 1X TAE 1.7% agarse gel. The linear range f Nrgen s C. cladspriides PCR Detectin Kit has been determined t cver cncentratins frm 100 fg t 1 ng Under the cnditins f the Nrgen s C. cladspriides DNA Islatin prcedure, Nrgen s C. cladspriides PCR Detectin Kit cvers a linear range frm 100 cpies t 1 x 10 6 cpies. Frequently Asked Questins 1. Hw many samples shuld be included per PCR run? Nrgen s C. cladspriides PCR Detectin Kit is designed t test 24 samples. Fr every 6 samples, a nn-template cntrl (Nuclease Free Water) and a Psitive Cntrl must be included. It is preferable t pl and test 6 samples at a time. If nt, the prvided Psitive Cntrl is enugh t run 3 samples at a time. 2. Hw can I interpret my results if neither the C. cladspriides PCR cntrl nr the C. cladspriides Islatin Cntrl (IsC) amplifies? If neither the C. cladspriides PCR cntrl nr the C. cladspriides Islatin Cntrl (IsC) amplifies, the sample must be re-tested. If the psitive cntrl shwed amplificatin, then the prblem ccurred during the islatin, where as if the Psitive cntrl did nt amplify, therefre the prblem has ccurred during the setup f the PCR assay reactin. 3. Hw shuld it be interpreted if nly the C. cladspriides PCR cntrl shwed amplificatin but neither the C. cladspriides target nr the C. cladspriides Islatin cntrl amplified fr a sample? This indicates a pr islatin. The islatin prcedure must be repeated. 4. Hw shuld it be interpreted if nly the C. cladspriides Islatin Cntrl (IsC) was amplified in a sample? The sample tested can be cnsidered as C. cladspriides negative. 5. Hw shuld it be interpreted if the C. cladspriides PCR cntrl and the C. cladspriides target shwed amplificatin in a sample? The sample tested can be cnsidered psitive. It culd happen when t much template was added t the reactin. 6. Hw shuld it be interpreted if nly the C. cladspriides target and the C. cladspriides PCR cntrl were amplified in a sample? The sample tested can be cnsidered as C. cladspriides psitive. 7. Hw shuld it be interpreted if nly the C. cladspriides target was amplified in a sample? It is recmmended that the islatin is repeated. 8. Hw shuld it be interpreted if nly the C. cladspriides PCR cntrl and the C. cladspriides Islatin cntrl shwed amplificatin in a sample? The sample tested can be cnsidered negative

10 9. What if I frgt t d a dry spin after my third wash? Yur first DNA elutin will be cntaminated with the Wash Slutin. This may dilute the DNA yield in yur first elutin and it may interfere with the PCR detectin, as ethanl is knwn t be a PCR inhibitr. 10. What if I frgt t add the C. cladspriides Islatin Cntrl (IsC) during the islatin? It is recmmended that the islatin is repeated. 11. What if I frgt t run the Cntrl RT-PCR fr the sample and I nly ran the Detectin RT- PCR and I btained a psitive result? The result can be cnsidered psitive. Hwever, any negative result must be verified by running the assciated cntrl PCR t ensure that it is a true negative and nt a false negative due t prblems with the RNA islatin r the PCR reactins. Related Prducts Prduct # Fungi/Yeast Genmic DNA Islatin kit Bacterial Genmic DNA Islatin Kit Plant/Fungi DNA Islatin Kit Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theretical expertise in sample and assay technlgies and the use f NORGEN prducts. If yu have any questins r experience any difficulties regarding Cladsprium cladspriides PCR Detectin Kit r NORGEN prducts in general, please d nt hesitate t cntact us. NORGEN custmers are a valuable surce f infrmatin regarding advanced r specialized uses f ur prducts. This infrmatin is helpful t ther scientists as well as t the researchers at NORGEN. We therefre encurage yu t cntact us if yu have any suggestins abut prduct perfrmance r new applicatins and techniques. Fr technical assistance and mre infrmatin, please cntact ur Technical Supprt Team between the hurs f 8:30 and 5:30 (Eastern Standard Time) at (905) r Tll Free at r call ne f the NORGEN lcal distributrs ( r thrugh at techsupprt@nrgenbitek.cm Schmn Parkway, Thrld, ON Canada L2V 4Y6 Phne: (905) Fax: (905) Tll Free in Nrth America: Nrgen Bitek Crp. PI