RIDA GENE Pneumocystis jirovecii realtime PCR Art. Nr.: PG1905 100 Reactions For invitro diagnostic use. 20 C RBiopharm AG, An der neuen Bergstraße 17, D64297 Darmstadt, Germany Tel.: +49 (0) 61 51 81 020 / Telefax: +49 (0) 61 51 81 0220
1. Intended use For in vitro diagnostic use. RIDA GENE Pneumocystis jirovecii is a realtime PCR for the direct qualitative and quantitative detection of Pneumocystis jirovecii from human bronchoalveolar lavage fluid (BAL). 1,2 The RIDA GENE Pneumocystis jirovecii realtime PCR is intended to use as an aid in diagnosis for respiratory infections caused by Pneumocystis jirovecii. 2. Explanation of the test Pneumocystis jirovecii (former P. carinii) belongs to the family of Pneumocystidaceae and may lead to an interstitial pneumonia. Opportunistic infections are a major problem in immunocompromised patients, for example HIV/AIDS patients, chemotherapytreated patients and patients receiving an organ transplant. Pneumocystis jirovecii causes respiratory infections and is the most common opportunistic illness in HIVinfected people. Pneumocystis jirovecii does not cause any harm in healthy people and is widely spread among the normal population. However, immunocompromised people infected with Pneumocystis jirovecii, develop pneumonia with symptoms including dry cough, shortness of breath, tachypnoe and fever. 3 Although HAART therapy decreased the Pneumocystis jirovecii incidence by 3.4 % per year after 1996, it is estimated that still 9 % among hospitalized HIV/AIDS patients and 1 % among solid organ transplant recipients are infected. 4 According to the Center for disease control (CDC), Pneumocystis jirovecii causes 100 % mortality in patients without treatment and the mortality rate in immunocompromised patients is between 5 % 40 % in treated patients. 4 The mortality from Pneumocystis jirovecii in HIVuninfected patients can be as high as 40 %. 5 Until now, detection of Pneumocystis jirovecii was done by immunofluorescence staining. However, due to its low sensitivity, this is now substituted by PCR. 2 3. Test principle RIDA GENE Pneumocystis jirovecii is a realtime PCR for the direct, qualitative and quantitative detection Pneumocystis jirovecii from human bronchoalveolar lavage fluid (BAL). After DNA isolation, amplification of the gene fragment (if present) specific for Pneumocystis jirovecii (LSU; large subunit) occurs. The amplified targets are detected with hydrolysis probes, which are labeled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore). In the presence of a target the probes hybridize to the amplicons. During the extension step, the Taqpolymerase breaks the reporterquencher proximity. The reporter emits a fluorescent signal, which is detected by the optical unit of a realtime PCR instrument. The fluorescence signal increases with the amount of formed amplicons. With the 2 RIDA GENE Pneumocystis jirovecii 130930
Standard DNA A, B and C included in the kit, it is possible to quantify the results. The RIDA GENE Pneumocystis jirovecii realtime PCR kit contains an internal control (ICD) that detects PCR inhibition, monitors reagent integrity and confirms that nucleic acid extraction was sufficient. 4. Reagents provided Tab.1: Reagents provided (Reagents provided in the kit are sufficient for 100 determinations) Kit Code Reagent Amount Lid Color 1 Reaction Mix 2x 1100 µl yellow 2 TaqPolymerase 1x 11 µl red D Internal Control DNA 2x 1800 µl orange N PCR Water 1x 500 µl white P Positive Control 1x 200 µl blue A Standard DNA A 1x 100 µl dark blue B Standard DNA B 1x 100 µl dark blue C Standard DNA C 1x 100 µl dark blue 5. Storage instructions Protect all reagents from light and store at 20 C. All reagents can be used until the expiration date. After expiry the quality guarantee is no longer valid. Carefully thaw reagents before using (e.g. in a refrigerator at 2 8 C). Reagents can sustain up to 20 freeze/thaw cycles without influencing the assay performance (e.g. after the first thawing separate it in aliquots and freeze immediately). During PCR preparation all the reagents should be stored cold in an appropriate way (2 8 C). 6. Additional equipment and materials required DNAExtraction system for bronchoalveolar lavage fluid (BAL) (e.g. NucliSENS easy MAG (biomérieux), MagNA Pure 96 (Roche)) RIDA GENE Pneumocystis jirovecii 130930 3
Realtime PCR instrument: Roche: LightCycler 1.5, LightCycler 2.0, LightCycler 480II Cepheid: SmartCycler Applied Biosystems: ABI 7500 Abbott: m2000rt Stratagene: Mx3005P QIAGEN: RotorGene Q BioRad: CFX96 RIDA GENE Color Compensation Kit II (PG0002) to run the LightCycler 1.5 and 2.0 (Roche) Realtime PCR consumables (plates, tubes, foil) Centrifuge with a rotor for the reaction vials Vortexer Pipettes (0.5 20 µl, 20 200 µl, 100 1000 µl) Filter tips Powderfree disposal gloves 7. Precautions for users For in vitro diagnostic use only. Extraction, PCR preparation and the PCR run should be separated in different rooms to avoid crosscontaminations. This test must only be performed by laboratory personnel trained in molecular biology methods. Strictly follow the working instructions. When handling samples, wear disposable gloves. After finishing the test, wash your hands. Do not smoke, eat or drink in areas, where samples or test reagents are being used. Samples must be treated as potentially infectious, as well as all reagents and materials being exposed to the samples, and have to be handled according to the national safety regulations. Do not use the kit after the expiration date. 8. Test procedure 8.1 DNA Isolation from bronchoalveolar lavage 4 RIDA GENE Pneumocystis jirovecii 130930
To isolate DNA from bronchoalveolar lavage, we recommend using a commercially available DNA extraction system (e.g. NucliSENS easy MAG (biomérieux)). Isolate DNA according to manufacturer s instructions. The RIDA GENE Pneumocystis jirovecii realtime PCR kit contains an internal control (ICD) that detects PCR inhibition, monitors reagent integrity and confirms that nucleic acid extraction was sufficient. If the ICD is used as an extraction control for the sample preparation procedure and as PCR inhibition control, 20 µl of the ICD has to be added during extraction procedure. The ICD should always be added to the specimenlysis buffer mixture and must not be added directly to the specimen. If the ICD is used only as a PCR inhibition control, 1 µl of the ICD should be added to the MasterMix (see Tab.3). 8.2 MasterMix preparation Calculate the total number of PCR reactions (sample and control reactions) needed. One positive control and negative control must be included in each assay run. We recommend calculating an additional volume of 10% to compensate imprecise pipetting (see Tab.2, Tab.3). Thaw, mix gently and briefly centrifuge the Reaction Mix, the TaqPolymerase, the Positive Control, the PCR Water and the ICD before using. Keep reagents appropriately cold during working step (2 8 C). Tab.2: Calculation and pipetting example for 10 reactions of the MasterMix (ICD as extraction and PCR inhibition control) Kit code MasterMix components Volume per reaction 10 reactions (10 % extra) 1 Reaction Mix 19.9 µl 218.9 µl 2 TaqPolymerase 0.1 µl 1.1 µl Total 20.0 µl 220 µl Mix the components of the MasterMix gently and briefly spin down. Tab.3: Calculation and pipetting example for 10 reactions of the MasterMix (ICD only as PCR inhibition control) Kit Code MasterMix components Volume per reaction 10 reactions RIDA GENE Pneumocystis jirovecii 130930 5
(10 % extra) 1 Reaction Mix 19.9 µl 218.9 µl 2 TaqPolymerase 0.1 µl 1.1 µl D Internal Control DNA 1.0 µl 11 µl Total 21.0 µl 231.0 µl Mix the components of the MasterMix gently and briefly spin down. 8.3 Preparation of the PCRMix Pipette 20 µl of the MasterMix in each reaction vial (tube or plate). Negative control: Add 5 µl PCR Water as negative control to the prepipetted MasterMix. Note: If the ICD is used as extraction control for the sample preparation procedure and as PCR inhibition control, we recommend to add 1 µl of the ICD to the negative control PCR Mix. Sample: Add 5 µl DNAExtract to the prepipetted MasterMix. Positive control: Add 5 µl Positive Control to the prepipetted MasterMix. Note: If the ICD is used as extraction control for the sample preparation procedure and as PCR inhibition control, we recommend to add 1 µl of the ICD to the positive control PCRMix. Standard DNA (A, B, C): Add 5 µl Standard DNA (A, B, C) to the prepipetted MasterMix in the designated reaction tubes. Note: Using the following cyclers requires to include a standard curve in each run: SmartCycler (Cepheid, closed system), ABI 7500 (Applied Biosystems), m2000rt (Abbott) and CFX96 (BioRad) For all other cyclers, only one sample of the standard curve has to be included in the experimental setup as calibrator for each new realtime PCR run. Here, the application of a standard curve is only required to run once per lot number. Cover tubes or plate. Spin down and place in the realtime PCR instrument. The PCR reaction should be started according to the PCR instrument Setup (see Tab.4, Tab. 5). 6 RIDA GENE Pneumocystis jirovecii 130930
8.5 PCR Instrument Setup Tab.4: Realtime PCR profile for LightCycler 1.5, LightCycler 2.0, LightCycler 480II, SmartCycler and RotorGene Q Initial Denaturation 1 min, 95 C Cycles PCR Denaturation Annealing/Extension 45 Cycles 10 sec, 95 C 15 sec, 60 C Temperature Transition Rate / Ramp Rate Maximum Note: Annealing and Extension occur in the same step. Note: Check that the Manual Thresh Fluor Units for Channel 1 is set to 30.0 and for Channel 2 is set to 5.0 on the SmartCycler (Cepheid). Due to variations between different cyclers, it may be required to individually adapt the Manual Thresh Fluor units for channel 1. Tab.5: Realtime PCR profile for Mx3005P, ABI 7500, m2000rt and CFX96 Initial Denaturation 1 min, 95 C Cycles PCR Denaturation Annealing/Extension 45 Cycles 15 sec, 95 C 30 sec, 60 C Temperature Transition Rate / Ramp Rate Maximum Note: Annealing and Extension occur in the same step. Note: The total copy number per reaction of Standard DNA A, B and C has to be typed in into the Setup File of the software programme of the respective realtime PCR cycler. A total volume of 5 µl DNA is used resulting in following concentrations: Standard DNA A: Standard DNA B: Standard DNA C: 5 x 10 1 copies/reaction 5 x 10 3 copies/reaction 5 x 10 5 copies/reaction Note: The standard curve can be saved on the realtime PCR cycler for each parameter. Apart from the SmartCycler (Cepheid, closed system), the ABI 7500 (Applied Biosystems), the m2000rt (Abbott) and the CFX96 (BioRad) cycler, the standard RIDA GENE Pneumocystis jirovecii 130930 7
curve is only required to run once per lot number. Using the SmartCycler (Cepheid, closed system), the ABI 7500 (Applied Biosystems), the m2000rt (Abbott) and the CFX96 (BioRad) cycler requires to include a standard curve in each run. For all other cyclers, only one sample of the standard curve has to be included in the experimental setup as calibrator for each new realtime PCR run. 8.6 Detection channel Setup Tab.6: Selection of appropriate detection channels 8 RIDA GENE Pneumocystis jirovecii 130930
Realtime PCR Instrument Roche LightCycler 1.5 Roche LightCycler 2.0 Roche LightCycler 480II Cepheid SmartCycler ABI 7500 Detection Detection Channel Note Pneumocystis jirovecii F1 RIDA GENE Color Compensation Kit II (PG0002) ICD F2 is required Pneumocystis jirovecii 530 RIDA GENE Color Compensation Kit II (PG0002) ICD 560 is required Pneumocystis jirovecii 465/510 ICD 533/580 Pneumocystis jirovecii Kanal 1 ICD Kanal 2 RIDA GENE Color Compensation Kit Color Compensation kit is not required Check that the Manual Tresh Flour Units for Channel 1 is set on 30.0 and for Channel 2 is set on 5.0 Pneumocystis jirovecii FAM Check that passive reference option ROX ICD VIC is none Abbott m2000rt Pneumocystis jirovecii ICD FAM VIC Stratagene Mx3000P/ Mx3005P Pneumocystis jirovecii ICD FAM HEX Check that reference dye is none Qiagen RotorGene Q Pneumocystis jirovecii ICD Green Yellow BioRad CFX96 Pneumocystis jirovecii ICD FAM HEX 9. Result interpretation The analysis of the samples is done by the software of the used realtime PCR instrument according to the manufacturer` s instructions. Positive and negative controls have to show correct results (see Fig.1). Fig.1: Correct run of the positive and negative control (Pneumocystis jirovecii) on the RIDA GENE Pneumocystis jirovecii 130930 9
LightCycler 480II The result interpretation is done according to Table 7. Tab.7: Sample interpretation Detection of Pneumocystis jiroveciispecific target gene Pneumocystis jirovecii ICD Result positive positive/negative Pneumocystis jirovecii detectable negative positive Target gene not detectable negative negative Invalid Pneumocystis jirovecii is detected, if the sample DNA and the Internal Control DNA (ICD) show an amplification signal in the detection system. Pneumocystis jirovecii is also detected, if the sample DNA shows an amplification signal but none for the Internal Control DNA (ICD) in the detection system. The detection of the internal amplification control is not necessary because high 10 RIDA GENE Pneumocystis jirovecii 130930
concentrations of the amplicon can cause a weak or absent signal of the Internal Control DNA (ICD). Pneumocystis jirovecii is not detected, if the sample DNA shows no amplification signal, but an amplification signal for the Internal Control DNA (ICD) in the detection system. An inhibition of the PCR reaction can be excluded by the detection of the Internal Control DNA (ICD). A sample is invalid, if the sample DNA and Internal Control DNA (ICD) show no amplification signal in the detection system. The sample contains a PCR inhibitor. The extracted sample needs to be further diluted with PCR water (1:10) and reamplified, or the isolation and purification of the sample has to be improved. 9.1 Quantification of samples To quantify Pneumocystis jiroveciipositive samples, a standard curve with the Standard DNA A, B and C has to be performed separately. The standard curve measurement has to be saved separately. However, the same standard curve measurement can be used in all runs with products from the same lot number by importing the saved experiment. Note: This is not valid for the following cyclers: SmartCycler (Cepheid, closed system), ABI 7500 (Applied Biosystems), m2000rt (Abbott) and CFX96 (Bio Rad). Here, a standard curve hast to be included in each run. For all other cyclers, one sample of the standard curve has to be included in the experimental setup as calibrator for each new realtime PCR run. To quantify Pneumocystis jiroveciipositive samples, all Standard DNA samples (A, B and C), the positive and negative control as well as the unknown samples to be quantified, have to be selected and analyzed according to the instructions of the cycler manufacturer. Note: For further information on quantification of the samples please contact pcr@rbiopharm.de 10. Performance characteristics 10.1 Clinical Performance RIDA GENE Pneumocystis jirovecii 130930 11
In a retrospective clinical validation study we analyzed 154 extracted specimens (BAL) with the RIDA GENE Pneumocystis jirovecii assay and an inhouse realtime PCR assay in an institute in Germany. Tab.8 Correlation of the Pneuomocystis jirovecii results with the RIDA GENE Pneumocystis jirovecii realtime PCR and reference inhouse realtime PCR. Inhouse realtime PCR Positive Negative Total Comments RIDA GENE Pneumocystis jirovecii Positive 26 0 26 Sensitivity 83.9 % Negative 5 a) 123 128 Specificity 100 % a) Total 31 123 154 Five (5) samples are below the limit of the detection (LOD) of the RIDA GENE Pneumocystis jirovecii assay with a Cp value >35 in the reference inhouse realtime PCR assay. 10.2 Analytical sensitivity The RIDA GENE Pneumocystis jirovecii realtime PCR has a detection limit of 5 DNA copies per reaction for Pneumocystis jirovecii (s.fig.2). Fig.2: Dilution series Pneumocystis jirovecii (10 5 10 1 DNA copies per µl) on the LightCycler 480II 12 RIDA GENE Pneumocystis jirovecii 130930
The detection limit of the whole procedure depends on the sample matrix, DNAextraction and DNAconcentration. 10.3 Analytical specificity The RIDA GENE Pneumocystis jirovecii realtime PCR is specific for Pneumocystis jirovecii. No crossreaction could be detected for the following species (see Tab.9): Tab.9: Crossreactivity testing RIDA GENE Pneumocystis jirovecii 130930 13
Acinetobacter baumannii Strain 5377 Haemophilus influenzae Rd Human parainfluenza virus 2 strain Greer Neisseria meningitidis Strain FAM18 Adenovirus Zellkulturüberstand Herpes simplex virus 1 strain McIntyre Human parainfluenza virus 4b strain CH19503 Serratia liquefaciens Adenovirus 1, Human, strain Adenoid 71 Adenovirus 7, Human, Strain Gomen Bordetella parapertussis Strain 12822 Bordetella pertussis Tohama 1 Herpes simplex virus 2 strain MS Human respiratory syncitial virus strain Long Human respiratory syncitial virus strain 9320 Human parainfluenza virus 1 strain C35 Klebsiella oxytoca Staphylococcus aureus Klebsiella pneumoniae strain MGH78578 Legionella pneumophila subsp. Pneumophila Mycoplasma pneumoniae Strain FH of Eaton Agent Staphylococcus haemolyticus SM131 Streptococcus pneumoniae strain NCTC 7465 Acrobacter butzleri Campylobacter upsaliensis Cryptosporidium muris Proteus vulgaris Adenovirus 40, Human, Strain Dugan Candida albicans Cryptosporidium parvum Pseudomonas aeruginosa Adenovirus 41, Human, Strain Tak Citrobacter freundii E. coli (O26:H) Rotavirus Zellkultur Aeromonas hydrophila Citrobacter freundii NCTC 9750 E. coli (O6) Salmonella enteritidis Astrovirus Zellkultur Clostridium difficile E. coli (O157:H7) Shigella flexneri Bacillus cereus Clostridium perfringens Entamoeba histolytica Salmonella typhimurium Bacteroides fragilis Campylobacter coli Clostridium bifermentans Clostridium sporogenes Staphylococcus Enterobacter cloacae hominis subsp. novobiosepticus R22 Enterococcus faecalis Staphylococcus epidermidis Campylobacter jejuni Clostridium novyi Giardia intestinalis Portland 1 Vibrio parahaemolyticus Campylobacter fetus subsp. Fetus Clostridium septicum Giardia intestinalis WB Clone C6 Yersinia enterocolitica Campylobacter lari subsp. Lari Clostridium sordellii Giardia lamblia 11. Limitations of the method 1. The result of molecular analysis should not lead to the diagnosis, but always be considered in the context of medical history and symptoms of the patient. 2. This assay is only validated for human bronchoalveolar lavage fluid (BAL). 14 RIDA GENE Pneumocystis jirovecii 130930
3. Inappropriate specimen collection, transport, storage and processing or a pathogen load in the specimen below the analytical sensitivity can result in false negative results. 4. The presence of PCR inhibitors may cause invalid results. 5. Mutations or polymorphisms in primer or probe binding regions may affect detection of new variants resulting in a false negative result with the RIDA GENE Pneumocystis jirovecii assay. 6. As with all PCR based in vitro diagnostic tests, extremely low levels of target below the limit of detection (LoD) may be detected, but results may not be reproducible. 12. Literature 1. Linssen CF et al. Interlaboratory comparison of three different realtime PCR assays for the detection of Pneumocystis jirovecii in bronchoalveolar lavage fluid samples 2006, 55: 12291235. 2. Tia T et al. A highly sensitive novel PCR assay for the detection of Pneumocysits jirovecii DNA in bronchoalveolar lavage specimens from immunocompromised patients 2012, 18: 598603. 3. Borde JP et al. Aktuelle Diagnostik und Therapie der Pneumocystisjirovecii Pneuomonie. Dtsch Med Wochenschr 2011, 136: 14261430. 4. Centers for Disease Control and Prevention. Pneumocystis pneumonia Statistics 2012. 5. Krajicek BJ et al. Pneumocystis pneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin Chest Med 2009, 30: 265278. RIDA GENE Pneumocystis jirovecii 130930 15