RIDA GENE Viral Stool Panel II real-time RT-PCR

Transcription

1 RIDA GENE Viral Stool Panel II realtime RTPCR Art. No.: PG Reactions For invitro diagnostic use. 20 C RBiopharm AG, An der neuen Bergstraße 17, D64297 Darmstadt, Germany Tel.: +49 (0) / Telefax: +49 (0)

2 1. Intended use For in vitro diagnostic use. RIDA GENE Viral Stool Panel II is a multiplex realtime RTPCR for the direct, qualitative detection and differentiation of rotavirus, adenovirus and astrovirus in human stool samples. 1,2,3 The RIDA GENE Viral Stool Panel II multiplex realtime RTPCR is intended for use as an aid in diagnosis of gastroenteritis caused by rotavirus, adenovirus and astrovirus, respectively. 2. Explanation of the test Acute Gastroenteritis is one of the main causes of morbidity and mortality worldwide. Especially in children, enteral viruses are the primary cause of gastroenteritis. In the US, viral infections cause approximately 30.8 million cases of gastroenteritis, yearly. 4 The most important pathogens causing diarrhea are rotavirus, adenovirus and astrovirus. Rotaviruses belong to the Reoviridae familiy of nonenveloped icosahedral doublestranded RNA (dsrna) viruses. Symptoms of rotavirus infection are usually vomiting, watery diarrhoea and abdominal pain. The virus is transmitted by the fecaloral route through contaminated hands and objects. Rotavirus is the main cause of diarrhoea in children aged under five and is responsible for the death of an estimated 611,000 children worldwide each year. 5 Adenoviruses belong to the Adenoviridae family of nonenveloped icosahedral doublestranded (dsdna) viruses. One differentiates 51 serotypes of human adenoviruses and they are classified into six groups (A F). Adenoviruses mainly cause respiratory diseases, whereas Gastroenteritis is primarily caused by serotype 40 and The serotypes 31, 12, 18, 1, 2, 5 and 6 are less often associated with acute diarrhoea. 7 Astroviruses are singlestranded (ssrna) viruses and belong to the family of Astroviridae. An astroviraldependent Gastroenteritis is primarily manifested by dirarrhoea, but can also be accompanied by vomiting and fever. In developed countries, the astrovirus incidence is between 2 9%, where the disease mainly affects children under the age of two. 8 Today, there are 8 serotypes described, with serotypes 15 being most relevant. The infection is transmitted by contaminated foods, by water and by the fecaloral route. 1. Test principle The RIDA GENE Viral Stool Panel II assay is a multiplex realtime PCR for the direct, qualitative detection and differentiation of rotavirus, adenovirus and astrovirus. After RNAisolation, amplification of the gene fragments specific for rotavirus (NSP3), 2 RIDA GENE Viral Stool Panel II

3 adenovirus (Hexon) and astrovirus (CAP; capsid protein) occurs, if present. 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. The RIDA GENE Viral Stool Panel II assay contains an Internal Control RNA (ICR) as an internal control of sample preparation procedure and to determine possible PCRinhibition. 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 700 µl yellow 2 PPMix 1x 770 µl green 3 Enzyme Mix 1x 80 µl red R Internal Control RNA 2x 1800 µl brown N PCR Water 1x 500 µl white P Positive Control 1x 100 µl 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 5 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). RIDA GENE Viral Stool Panel II

4 6. Additional equipment and materials required RNAExtraction kit for stool samples (e.g. RIDA Xtract) or RNAExtraction system for stool samples (e.g. Maxwell 16 (Promega)) Realtime PCR instrument: Roche: LightCycler 480II Cepheid: SmartCycler Applied Biosystems: ABI 7500 Abbott: m2000rt Stratagene: Mx3005P QIAGEN: RotorGene Q BioRad: CFX96 Note: Use on the RotorGene Q (QIAGEN) only 0.1 ml tubes. RIDA GENE Color Compensation Kit I (PG0001) to run the LightCycler 480II (Roche) Realtime PCR consumables (plates, tubes, foil) Centrifuge with a rotor for the reaction vials Vortexer Pipettes ( µl, µl, 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. 4 RIDA GENE Viral Stool Panel II

5 8. Test procedure 8.1 Sample Preparation RNA isolation from Stool Samples For RNA isolation of human stool samples use a commercially available RNA extraction kit (e.g. RIDA Xtract) or RNA extraction system (e.g. Maxwell 16 (Promega))). Extract viral RNA according to the manufacturer s instructions. We recommend to dilute the stool sample before extraction 1:10 with water. Vortex intensely and centrifuge at 12,000 rpm for 1 min. Use from the supernatant an appropriate volume according to the manufacturer s instruction. The RIDA GENE Viral Stool Panel II assay contains an Internal Control RNA (ICR), which can either be used as PCR inhibition control or as extraction control for the sample preparation procedure and as a PCR inhibition control. If the ICR is used only as a PCR inhibition control, 1µl of the ICR should be added to the Master Mix (s. Tab.3). If the ICR is used as an extraction control for the sample preparation procedure and as PCR inhibition control, 20 µl of the ICR has to be added during extraction procedure. The ICR should always be added to the specimenlysis buffer mixture and must not be added directly to the specimen. 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 to calculate an additional volume of 10 % to compensate imprecise pipetting (see Tab.2, Tab.3). Thaw, mix gently and centrifuge briefly the Reaction Mix, the PPMix, the Positive Control, the PCR Water and the ICR before using. Keep reagents appropriately cold during working step (2 8 C). Tab.2: Calculation and pipetting example for 10 reactions of the Master Mix (ICR as extraction and PCR inhibition control) Kit code MasterMix components Volume per reaction 10 reactions (10 % extra) 1 Reaction Mix 12.5 µl µl 2 PPMix (PrimerProbeMix) 6.9 µl 75.9 µl 3 Enzyme Mix 0.7 µl 7.7 µl Total 20.1 µl µl Mix the components of the Master Mix gently and briefly spin down. RIDA GENE Viral Stool Panel II

6 Tab.3: Calculation and pipetting example for 10 reactions of the Master Mix (ICR only as PCR inhibition control) Kit Code MasterMix components Volume per reaction 10 reactions (10 % extra) 1 Reaction Mix 12.5 µl µl 2 PPMix (PrimerProbeMix) 6.9 µl 75.9 µl 3 Enzyme Mix 0.7 µl 7.7 µl R Internal Control RNA 1.0 µl 11 µl Total 21.1 µl µl Mix the components of the Master Mix gently and briefly spin down. 8.3 Preparation of the RTPCR Mix 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 ICR is used as extraction control for the sample preparation procedure and as PCR inhibition control, we recommend to add 1 µl of the ICR to the negative control RTPCR Mix. Sample: Positive control: Add 5 µl RNAExtract to the prepipetted MasterMix. Add 5 µl Positive Control to the prepipetted MasterMix. Note: If the ICR is used as extraction control for the sample preparation procedure and as PCR inhibition control, we recommend to add 1 µl of the ICR to the positive control RTPCR Mix. Cover tubes or plate. Spin down and place in the realtime PCR instrument. The RTPCR reaction should be started according to the PCR instrument Setup (see Tab.4). 6 RIDA GENE Viral Stool Panel II

7 8.4 PCR Instrument Setup Tab.4: Realtime RTPCR profile Reverse Transcription Initial Denaturation 10 min, 58 C 1 min, 95 C Cycles PCR Denaturation Annealing/Extension 45 Cycles 15 sec, 95 C 30 sec, 55 C Temperature Transition Rate / Ramp Rate Maximum Note: Annealing and Extension occur in the same step Note: Check that the Manual Thres. Fluor Units for Channel 1 is set to 30.0 and for Channel 2 to 4 is set to 5.0 on the SmartCycler (Cepheid). Due to variations between different cyclers, it may be required to individually adapt the Manual Thres. Fluor Units for channel 1. RIDA GENE Viral Stool Panel II

8 8.5. Detection Channel Setup Tab.6: Selection of appropriate detection channels Realtime PCR Instrument Detection Detection channel Note Roche LightCycler 480II Cepheid SmartCycler Rotavirus 465/510 ICR 533/580 Astrovirus 533/610 Adenovirus 618/660 Rotavirus Kanal 1 ICR Kanal 2 Astrovirus Kanal 3 Adenovirus Kanal 4 RIDA GENE Color Compensation Kit I (PG0001) is required Check that the Manual Thres. Fluor Units for Channel 1 is set on 30.0 and for Channel 2 to 4 is set to 5.0 Rotavirus FAM ABI 7500 ICR Astrovirus VIC ROX Check that passive reference option ROX is none Adenovirus Cy5 Rotavirus FAM Abbott m2000rt ICR Astrovirus VIC ROX Adenovirus Cy5 Rotavirus FAM Stratagene Mx3005P ICR Astrovirus HEX ROX Check that the reference dye is none Adenovirus Cy5 Rotavirus Green Qiagen RotorGene Q ICR Astrovirus Yellow Orange The gain settings have to be set to 5, according to the default settings Adenovirus Red Rotavirus FAM BioRad CFX96 ICR Astrovirus VIC ROX Adenovirus Cy5 8 RIDA GENE Viral Stool Panel II

9 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.2, Fig.3). The positive control has a concentration of 10 3 copies/µl. In each PCR run it is used in a total amount of 5 x 10 3 copies. Fig.1: Correct run of the positive and negative control (rotavirus) on the LightCycler 480II Fig.2: Correct run of the positive and negative control (adenovirus) on the LightCycler 480II RIDA GENE Viral Stool Panel II

10 Fig.3: Correct run of the positive and negative control (astrovirus) on the LightCycler 480II The result interpretation is done according to Table 7. Tab.7: Sample interpretation Target genes Rotavirus Adenovirus Astrovirus ICD Result positive negative negative positive/negative Rotavirus negative positive negative positive/negative Adenovirus negative negative positive positive/negative Astrovirus negative negative negative positive Negative (Target genes not detectable) negative negative negative negative Not evaluable A sample is evaluated negative, if the sample shows no amplification signal in the detection system, but the Internal Control RNA (ICR) is positive. An inhibition of the PCR reaction or a failure in the extraction procedure can be excluded by the detection of the Internal Control RNA (ICR). 10 RIDA GENE Viral Stool Panel II

11 A sample is evaluated positive, if both, the sample and the Internal Control RNA, (ICR) show an amplification signal in the detection system. A sample is evaluated positive, if the sample shows an amplification signal in the detection system, but the Internal Control RNA (ICR) is negative. The detection of the internal amplification control is not necessary, because high concentrations of the amplicon can cause a weak or absent signal of the internal amplification control. A sample is evaluated invalid, if both, the sample and the Internal Control RNA (ICR) show no amplification signal in the detection system. The sample contained a PCR inhibitor or a failure occurred in the extraction procedure. 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. 10. Test characteristics 10.1 Analytical sensitivity The RIDA GENE Viral Stool Panel II realtime RTPCR has a detection limit of 50 RNA copies per reaction and a detection limit of 50 DNA copies per reaction, respectively (see Fig.4, Fig.5, Fig.6). Fig.4: Dilution series rotavirus ( RNA copies per µl) on the LightCycler 480II RIDA GENE Viral Stool Panel II

12 Fig.5: Dilution series adenovirus ( RNA copies per µl) on the LightCycler 480II Fig.6: Dilution series astrovirus ( RNA copies per µl) on the LightCycler 480II The detection limit of the whole procedure depends on the sample matrix, RNAextraction and RNAconcentration. 12 RIDA GENE Viral Stool Panel II

13 10.2 Analytical specificity The analytical specificity of the RIDA GENE Viral Stool Panel II multiplex realtime PCR is specific for rotavirus, adenovirus and astrovirus. No crossreaction could be detected for the following species (see Tab.8): Tab.8: Crossreactivity testing Arcobacter butzleri Clostridium bifermentans Enterobacter cloacae Serratia liquefaciens Aeromonas hydrophila Clostridium difficile Enterococcus faecalis Shigella flexneri Bacillus cereus Clostridium sporogenes Giardia lamblia Staphylococcus aureus Bacteroides fragilis Clostridium septicum Giardia intestinalis Portland 1 Staphylococcus epidermidis Campylobacter coli Clostridium novyi Giardia intestinalis WB Clone C6 Vibrio parahaemolyticus Campylobacter jejuni Clostridium sordellii Klebsiella oxytoca Yersinia enterocolitica Campylobacter fetus subsp. Fetus Cryptosporidium parvum Norovirus GGI Campylobacter lari subsp. Lari Cryptosporidium muris Norovirus GGII Campylobacter upsaliensis E. coli (O26:H) Proteus vulgaris Candida albicans E. coli (O6) Citrobacter freundii E. coli (O157:H7) Clostridium perfringens Entamoeba histolytica Pseudomonas aeruginosa Salmonella enteritidis Salmonella typhimurium RIDA GENE Viral Stool Panel II

14 10.3 Analytical reactivity The reactivity of the RIDA GENE Viral Stool Panel II multiplex realtime PCR was evaluated against previously positive characterized rotavirus, adenovirus and astrovirus samples (see Tab. 9). All tested viruses were detected by the RIDA GENE Viral Stool Panel II multiplex realtime PCR. Tab.9: Analytical reactivity testing (number of samples tested) Rotavirus (16) + Adenovirus (15) + Astrovirus (16) 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. The RIDA GENE Viral Stool Panel II assay is only validated for stool samples. 3. Inappropriate specimen collection, transport, storage and processing or a viral 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 new variants resulting in a false negative result with the RIDA GENE Viral Stool Panel II 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. 14 RIDA GENE Viral Stool Panel II

15 12. Literature 1. Pang XL, et al. Increased Detection of Rotavirus Using a Real Time Reverse TranscriptionPolymerase Chain Reaction (RTPCR) Assay in Stool Specimens From Children With Diarrhea. Journal of Medical Virology 2004, 72: Heim A, et al. Rapid and Quantitative Detection of Human Adenovirus DNA by RealTime PCR. Journal of Medical Virology 2003, 10: Kapoor A et al., Multiple novel Astrovirus species in human stool. Journal of general Virology 2009, 90: Mead PS, et al. EID 1999, 5: Parashar UD, et al. Rotavirus and Severe Childhood Diarrhea. Emerging Infectious Diseases 2006, 12: Robert Koch Institut. Keratoconjunctivitis epidemica und andere Konjunktivitiden durch Adenoviren. RKIRatgeber Infektionskrankheiten Merkblätter für Ärzte Wilhelmi I, et al. Viruses causing gastroenteritis. Clinical Microbiology and Infection 2003, 9: Guix S, et al. Human astrovirus diagnosis and typing: current and future Prospects. Letters of Applied Microbiology 2005, 41: RIDA GENE Viral Stool Panel II