Cepheid Genexpert Manual
BackgroundIn Kenya, access to early infant diagnosis and viral load monitoring services for HIV patients on ART is significantly hampered by sample transportation challenges and long turnaround times. Near patient care testing technologies have the potential to obviate such constraints. The Cepheid GeneXpert was launched in 2010 as a TB assay and in 2014 as a potential point of care HIV viral load assay. Whereas it is widely is used for TB in Kenya, its utility for HIV testing has not been evaluated. MethodsThis was a cross sectional study among 911 HIV Exposed infants and 310 HIV positive adults. Existing machines used for routine TB diagnosis were used in this study. The diagnostic accuracy of the qualitative assay was assessed using Roche CAP/CTM while the quantitative assay was assessed using with Abbott m2000 as the reference assays respectively.
Statistical analysis was done using Stata/MP Version 14 for Mac. Concordance values and misclassification were calculated at the clinical cutoff of 1000 cp/ml. ResultsThe sensitivity, specificity and accuracy of the GeneXpert HIV-1 qualitative assay were 99.23% (95% CI 97.24–99.90%), 98.91% (95% CI 97.76–99.55%) and 99.00% respectively.
For the quantitative assay, they were 92.50% (95% CI 79.61–98.43%), 100.00% and 97.00% respectively. All 30 (100%) users reported that the GeneXpert machine was easy to use, workflow was simple and TB diagnosis was not negatively affected.
In our hands, the median turn-around time for an individual qualitative and quantitative test was 90 minutes. A total of 58 (4.34%) errors and 28 (2.10%) invalid outcomes were experienced; 44 (3.29%) tests did not run to completion due to power outages. ConclusionGeneXpert HIV-1 qualitative and quantitative assay is an accurate test for the diagnosis of HIV in infants and for viral load monitoring. At the point of care, the GeneXpert machine’s simple work flow, ease of use and short test turnaround time present the potential to improve access to HIV testing and viral load monitoring. To integrate HIV diagnosis into the existing GeneXpert platforms for TB Diagnosis, there is need to scale up the infrastructure and to change the way work is done.
Citation: Bwana P, Ageng’o J, Mwau M (2019) Performance and usability of Cepheid GeneXpert HIV-1 qualitative and quantitative assay in Kenya. PLoS ONE 14(3):e0213865.Nei-yuan Hsiao, University of Cape Town Faculty of Health Sciences, SOUTH AFRICAReceived: October 31, 2018; Accepted: March 1, 2019; Published: March 22, 2019Copyright: © 2019 Bwana et al. This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All data are within the manuscript.Funding: This work was supported by Kenya Medical Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.
IntroductionBy 2015, 1.5 million (4.1%) of the 36.7 million people living with HIV worldwide were Kenyan,. Both early infant diagnosis and viral load testing, which are essential for monitoring HIV treatment care and are critical to achieving the UNAIDS 90-90-90 targets by 2020. Kenya has managed to reduce the prevalence of HIV from 7.4% in 2007 to 5.9% in 2015. Highly active antiretroviral therapy (HAART) is widely accessible and by 2017, treatment coverage was at 75%.Despite having 9 reference HIV testing labs, EID and VL testing coverage is only around 76% and 98% respectively. Sample transportation over long difficult roads and prolonged turnaround time to results hamper wider access to these services,.
Although the hub and spoke modelhas been implemented to improve sample networking, the hubs themselves are not testing labs and the turnaround time still remains a challenge. Loss to follow up is significant.
Near patient and point of care testing technologies have the potential to obviate these challenges.WHO recently approved several point of care assays for HIV diagnostics, including Cepheid Xpert HIV-1 Qual for HIV qualitative testingusing whole blood or dried blood spots (DBS) and Cepheid XpertHIV-1 Viral Load for HIV quantitative testing – using plasma, both using one fully integrated cartridge. These near patient assays, have reported encouraging performance in recent studies,. Among other test platforms still undergoing development, is the GeneXpert Omni, a portable, battery-operated, wireless, and web-enabled diagnostic test that can transmit test and instrument information in real time.In Kenya, both Alere q HIV 1/2 Detect and Xpert HIV-1 qualitative assays have been approved for Early Infant diagnosis. Cepheid Xpert MTB/RIF is also widely used for TB diagnosis.
However, neither Alere q HIV 1/2 Viral Load nor Cepheid Xpert HIV-1 Viral Load has been approved for viral load quantification. Additionally, the utility of already existing GeneXpert platforms for HIV testing alongside TB diagnostics has not been evaluated.This evaluation was intended to determine the accuracy and ease of use of the Xpert HIV-1 qualitative and quantitative assay (Cepheid, Sunnyvale, California, United States) in field evaluations. Study designAll the GeneXpert machines used for this study were already in situ and routinely used for TB diagnosis. At study inception, it was noted that all machines were in use during the day for TB diagnosis, since all the facilities that had them were hubs within a testing network.
It was determined that HIV testing was best done beginning late afternoon. Study personnel, though experienced users, were trained for this purpose.
Both the qualitative and the quantitative studies of the performance of the GeneXpert platform were cross sectional evaluations of samples obtained from facilities across the country. The usability data was collected through structured questionnaires.In order to assess the performance of the Xpert HIV-1 quantitative assay on GeneXpert platform, 100 plasma samples were tested on both the platform and on the Abbott m2000 assay and the results compared.To assess how different sample types performed against each other on the GeneXpert platform, 107 comparisons were made between plasma and whole blood, and 84 comparisons between plasma and DBS.To assess the qualitative performance of the Xpert HIV-1 qualitative assay, 911 infant DBS samples were tested against Roche CAP/CTM. HIV qualitative analysis.In field sites, two DBS filter papers were collected from infants as previously described. One of the filter papers was processed and tested on the GeneXpert platform, according to the manufacturer’s instructions. Briefly, from each filter paper, one dried blood spot was cut out and dropped into a vial containing proprietary buffer and incubated in an Eppendorf thermomixer for 15 minutes at 56° C while rotating at 500 rpm. The contents of the vial were then added into the Xpert HIV-1 Qual test cartridge and loaded onto the GeneXpert machine. Results were observed and recorded after 90 minutes.
The second DBS filter paper was shipped to the reference lab and tested on the Roche CAP/CTM platform according to manufacturer’s instructions as previously described. The results were recorded as HIV detected or HIV not detected. Quantitative performance evaluation.For viral load estimation on the GeneXpert platform a total of 1.2 ml of plasma was added into the Xpert HIV-1 Viral Load cartridge using a calibrated pipette.
The cartridge was closed and loaded onto the machine. Test results were observed and recorded after 90 minutes. The limit of detection was 40 cp/ml and the clinical cut off was 1000 cp/ml. For comparison, viral load estimation was done on the Abbott m2000 using plasma according to manufacturer’s instructions.
Briefly, 0.6ml of plasma was transferred into a reaction tube, which was then inserted into the Abbott m2000sp machine worktable. Automated sample extraction was conducted using m2000 version 2.0 open mode 0.6ml RNA HIV-1 assay protocol. The products were retrieved for RNA amplification and quantification, on the Abbott m2000rt platform. Test results were observed and recorded after 5 hours and the limit of detection was 40 cp/ml. The clinical cut off was 1000 cp/ml. Suitability of different sample types for use on GeneXpert platform.From each venous blood draw, whole blood, DBS and plasma was prepared and tested on GeneXpert platform.Plasma was processed as for performance evaluation.For DBS, one spot was cut out of the filter paper, dropped into an Eppendorf tube and 1.2 ml of 20 mM Tris HCl was added.
The tube was incubated in a thermo mixer for 15 minutes at 56° C while rotating at 500 rpm. All the contents of the Eppendorf tube were poured into the sample chamber of the Xpert HIV-1 Viral Load cartridge which was then loaded onto the machine. Test results were retrieved after 90 minutes. The limit of detection was 40 cp/ml and the clinical cut off was 1000 cp/ml.For whole blood, 0.75 ml of 20 mM Tris HCl buffer was first added onto the Xpert HIV-1 Viral Load cartridge. 100 μl of Whole blood sample was then added without delay. The cartridge was then handled according to the manufacturer’s instructions. The limit of detection was 40 cp/ml and the clinical cut off was 1000 cp/ml.
Usability evaluation.All study personnel received training on the assays being evaluated and were certified in their use. A structured questionnaire capturing steps to results, amount of waste generated, ease of use, errors and overall opinion of the technology was filled in by each personnel based on their experience on the machine. The usability data was collected by 30 GeneXpert platform users in Meru, Kakamega, Homa Bay and Siaya County referral Hospitals, as well as Yala, Pap Onditi, Ndhiwa and Kibera Sub County hospitals. Statistical analysisViral load data was transformed into log 10 cp/ml. Statistical analysis was performed using Stata/MP Version 14 for Mac. Accuracy was estimated using a 2 x 2 table at a cut off of 1000cp/ml.
Descriptive statistics were used to compare misclassification between platforms and sample types. Bland Altman comparison was conducted to assess the limits of agreement and the mean bias. Pearson Correlation Coefficient (r 2) was used to assess the linear fit for paired readings of log transformed viral load copies between platforms and between sample types. Simple descriptive statistics was used to report usability data. Performance characteristics of the GeneXpert quantitative assayThe concordance between the GeneXpert and Abbott m2000 assay at the clinical cut off of 1000cp/ml is depicted in.
All of the 37 samples testing 1000cp/ml on the Abbott m2000 assay tested 1000 cp/ml on the GeneXpert platform. Three of the 63 samples which tested 1000 cp/ml on the GeneXpert assay. Therefore, the overall concordance was 97.00% with an upward misclassification of 7.50%. There was 0% downward misclassification. Qualitative evaluation of GeneXpert HIV-1 assayA total of 911 HIV-exposed infants (HEI, median age = 1.5 months), were successfully recruited for the qualitative evaluation of GeneXpert HIV-1 Assay. A total of 901 DBS samples were successfully collected and tested on both the Xpert and Roche CAP/CTM platforms.The sensitivity of the Xpert HIV-1 Qual assay was 97.36% (95% CI 94.63–98.93%) while the specificity was 99.69% (95% CI 98.87–99.96%). The concordance was therefore 99.00%.
Below summarizes the performance characteristics of Xpert HIV -1 Qual assay. Usability characteristicsOf the 1555 qualitative and quantitative tests conducted on GeneXpert platform (including repeats and duplicates), 1436 (92.35%) tests passed. Sixteen (16, 1.03%) tests failed due to machine user errors, 38 (2.44%) due to hardware errors and 8 (0.51%) due to temperature. A further 28 (1.80%) tests gave invalid results while 29 (1.86%) tests failed due to power outage. The overall error rate was 7.65%.All the 30 (100%) users reported that the machine was easy to use, the work flow was simple, and the waste generated was little. There were five steps from sample collection to results.
The turn-around time for each test was approximately 90 minutes. The GeneXpert machine’s footprint was less than 4 square feet for the 16 module and less than 1 square foot for the 4 module. The machine was not rechargeable but had GSM capability.
DiscussionIn the attempt to achieve UNAIDS 90:90:90 goals, Kenya has come up with innovative approaches aimed at reaching underserved populations, including the hub and spoke model of laboratory networking, and the adoption of molecular point of care testing technologies.Recent WHO guidelines recommend technologies that deliver a “sample in/result out” service at the point of care. They should perform well using not only whole blood but also plasma and dried blood.
For HIV molecular diagnosis, only a few such technologies are available.Many countries have developed innovative ways of integrating HIV, TB and malaria programs in order to increase efficiency of resource usage and to improve the quality of health of patients. The GeneXpert platform is widely available in Kenya for TB diagnostics.
It can help integrate both TB and HIV diagnosis at the point of care, but before our study this potential had not been explored. In some countries, GeneXpert devices introduced for TB diagnostics are underutilized; that spare capacity can be leveraged for HIV diagnostics. GeneXpert is the primary tool for TB diagnostics in Kenya and in our study, we found that the machines were busy throughout the day, often dealing with backlogs. To prevent disruption to service delivery, we negotiated to test for HIV in the late afternoons and evenings.
We opine that health care workers who use GeneXpert for TB diagnosis at the current time would be amenable to testing for HIV using the same platform seamlessly if the national policy so required. Furthermore, if GeneXpert Omni proves to be similarly accurate in evaluations, then this technology has the potential to lower level health care facilities for service delivery. The impact of such interventions should be assessed periodically.In Kenya, the sample type of choice for early infant diagnosis is DBS.
Even in near patient settings, DBS remains an attractive solution, mainly due to the complexity of sample collection in infants and also for the reason that many facilities in rural Kenya have embraced the hub and spoke model for service delivery. Using DBS, the concordance of GeneXpert HIV-1 qualitative assay was 99.00%, a finding corroborated by other studies. This level of accuracy is very high and when viewed together with the challenges of transporting samples to central labs indicates that the GeneXpert qualitative assay has a significant role in EID testing in Kenya at the point of care.The GeneXpert quantitative assay performed poorly on dried blood spots in our hands.
However, for quantitative testing at the point of care and in near patient settings, DBS is an inconvenient sample type, and is unlikely to come into common usage, and therefore we consider the poor performance of the assay on DBS to be of minimal significance at the program level. The fact that the GeneXpert HIV-1 quantitative assay performed sub-optimally on whole blood is of much greater concern at the point of care, considering that blood is the easiest and most convenient sample type. The reason for this poor performance is unclear to us, but is supported by other studies.
However, when plasma samples were used instead, the GeneXpert HIV-1 quantitative assay was comparable in performance to the Abbott m2000 HIV-1/2. The preparation of plasma in most health facilities in Kenya is a routine activity, and the role of the assay in viral load testing at the point of care is therefore clear. That said, further assay optimization using whole blood will be necessary to minimize resource wastage.Current clinical guidelines use a cutoff of 1000 cp/ml for clinical decision-making. With plasma, a concordance of 97.00% was observed at that cutoff. Studies conducted on similar platforms and sample type have recorded varied performance with the concordance values ranging between 91.32%-100%–. Our findings also agree with those observed by WHO.In the GeneXpert qualitative assay, upward misclassification of 0.31% and downward misclassification of 2.64% was observed. This misclassification is quite low considering the intended use at the point of care.For the quantitative assay using plasma, upward misclassification was 3(3.00%) at the clinical cut off of 1000cp/ml.
There was no downward misclassification. This low misclassification rate compares favorably with other studies.
And indicates that GeneXpert can be used interchangeably with Abbott m2000 without anticipation of clinically relevant bias.When whole blood was compared with plasma on GeneXpert at a cut off of 1000cp/ml, 5 (4.67%) downward misclassifications and 12 (11.21%) upward misclassifications were observed. Clinically, this high upward misclassification rate would lead to more patients being identified as potential treatment failures, requiring adherence counseling and further plasma based VL testing for confirmation.When DBS was compared with plasma on GeneXpert at a cut off of 1000cp/ml, 31(36.90%) down misclassification was observed while upward misclassification was not reported. If DBS were to be used as the sample of choice, many patients would be would be identified as suppressed when they actually are not. This would unnecessarily expose them to complications related to high viral load. The suboptimal performance of whole blood and dried blood spots suggests that further optimization of the assay is necessary to improve performance. The positive bias of the quantitative GeneXpert assay has been reported in other studies.The overall test failure rate on the platform was 7.65%. This rate is comparable to similar studies.
A majority of errors were hardware related, at 2.44%, followed by errors due to power outage, at 1.86%. Of note, user errors were uncommon, at 1.03%.
This is important especially since user training can be capital intensive for some assays in the market. Temperature-related errors accounted for only 0.51% of all, implying that the technology is quite appropriate for sub-Saharan Africa, where temperatures can be as high as 40° C. Whereas errors experienced did not affect the overall performance of the respective assays, because all failed tests were repeated, they did lead to loss of cartridges and time, which are important considerations in resource limited settings. During testing, 28 (2.10%) tests gave invalid results on both the Xpert Qual and Viral Load assay. These invalid results may have been related to quality control during the manufacturing process or insufficient sample volumes. With strict adherence to good manufacturing practise by the manufacturer, and sufficient sample volumes, these errors may be reduced or eliminated altogether.The main strengths of this study lie in the fact that we assessed the usability and quantitative and qualitative performance of GeneXpert simultaneously in the context of existing platforms used for TB diagnostics.
Study limitationsThe most important limitation of this study was that data collection was not directly observed by the investigators, the quality of the work could not be guaranteed. However, the technicians were experienced and were well trained.The test devices did not have inbuilt power and went off during power outages.
This resulted in 44 (3.29%) tests not running to completion. There is a definite need for a power back up for use in the event of power outage to reduce wastage and turnaround time to results. ConclusionsIn this study, we have demonstrated that at the point of care, the GeneXpert assay shows excellent performance, has a simple work flow, is easy to use and has a short test turnaround time. Scale-up of the Xpert TB assay and the existing infrastructure and changing the way HIV and TB diagnostics work is done could serve as an implementation platform for the Xpert HIV VL and EID assays. Therefore, the GeneXpert assay can be used as a point of care assay for viral load estimation in resource limited settings. References.
1.UNAIDS. Fact Sheet 2015. World AIDs Day 2015 cited 2017 13 June. 2.National AIDS control council. Kenya AIDS response progress report.
2016. 3.UNAIDS. 90-90-90: An ambitious treatment target to help end the AIDS epidemic 2015. 4.National AIDS Control Council and the National AIDS and STD Control Programme. The Kenya 2007 HIV and AIDS Estimates and interim projected HIV prevalence and incidence trends for 2008 to 2015.
2009. 5.National AIDS and STD Control Programme (NASCOP). 2018. 6.Rouet F, Ekouevi DK, Chaix ML, Burgard M, Inwoley A, Tony TD, et al. Transfer and evaluation of an automated, low-cost real-time reverse transcription-PCR test for diagnosis and monitoring of human immunodeficiency virus type 1 infection in a West African resource-limited setting. J Clin Microbiol.
Pmid:15956387. 7.Rowley CF. Developments in CD4 and viral load monitoring in resource-limited settings.
Clin Infect Dis. Pmid:24218101. 8.Elrod JK, Fortenberry JL Jr. The hub-and-spoke organization design revisited: a lifeline for rural hospitals. BMC Health Serv Res.
2017;17(Suppl 4):795. Pmid:29297334. 9.Elrod JK, Fortenberry JL Jr.
The hub-and-spoke organization design: an avenue for serving patients well. BMC Health Serv Res. 2017;17(Suppl 1):457. Pmid:28722550. 10.Maria Buser RB, Jeff Lemaire. Point-of-Care Early Infant Diagnosis of HIV: Moving from Successful Field Evaluations to Routine Use. 2017.
11.WHO. Novel Point of Care tools for Early Infant Diagnosis of HIV. 2017. 12.Cepheid. Xpert ® HIV-1 Qual. 13.Ford N.
Viral load platforms for point of care testing and opportunities for TB/HIV integration. WHO, 2016. 14.Phillips AN, Cambiano V, Nakagawa F, Ford D, Apollo T, Murungu J, et al. Point-of-Care Viral Load Testing for Sub-Saharan Africa: Informing a Target Product Profile.
Open Forum Infect Dis. Pmid:27704016. 15.NOVITSKY V.
Genexpert Infinity Operator Manual
Point of Care testing;HIV-1 Viral load. 2015. 16.Cepheid. Xpert ® HIV-1 Viral Load. 17.WHO.
WHO Prequalification of In Vitro Diagnostics-Xpert ® HIV-1 Qual Assay. 2016. 18.WHO. WHO Prequalification of In Vitro Diagnostics-Xpert ® HIV-1 Viral Load with GeneXpert. 2017.
19.CHAI. Point of Care EID and VL Products: What’s in the Pipeline? 2016. 20.EGPAF. UNITAID/EGPAF Project to Optimize Early Infant HIV Diagnosis through the Introduction of Point of Care Testing. 2016. 21.USAID.
GeneXpert Omni Enables Increased Access to Accurate, Fast and Potentially Life-Saving Diagnosis for TB Patients 2017. 22.Sivapalasingam S, Ahmed A, Mendillo M, Holzman R, Marshed F, Mwamzuka M, et al.
Early detection of HIV infection among Kenyan infants using a reverse transcriptase activity assay. Pediatr Infect Dis J. Pmid:22581226. 23.Kageha S, Okoth V, Kadima S, Vihenda S, Okapesi E, Nyambura E, et al.
Discrepant test findings in early infant diagnosis of HIV in a national reference laboratory in Kenya: challenges and opportunities for programs. J Trop Pediatr. Pmid:22052701. 24.UNICEF A, CDC, CHAI, OGAC, EGPAF, LSHTM, ICAP, MSF, NHLS, USAID and WHO. Key considerations for introducing new HIV point-of-care diagnostic technologies in national health systems. 2018.
25.Ceffa S, Luhanga R, Andreotti M, Brambilla D, Erba F, Jere H, et al. Comparison of the Cepheid GeneXpert and Abbott M2000 HIV-1 real time molecular assays for monitoring HIV-1 viral load and detecting HIV-1 infection. J Virol Methods. Pmid:26709099. 26.Gous N, Scott L, Berrie L, Stevens W. Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert(R) HIV-1 Viral Load Assay. Pmid:27992495 technical support and Xpert HIV-1 VL testing kits for the study but was not involved in study design, data collection, analysis, or manuscript preparation.
This does not alter our adherence to PLOS ONE policies on sharing data and materials. 27.Gueudin M, Baron A, Alessandri-Gradt E, Lemee V, Mourez T, Etienne M, et al. Performance Evaluation of the New HIV-1 Quantification Assay, Xpert HIV-1 Viral Load, on a Wide Panel of HIV-1 Variants.
J Acquir Immune Defic Syndr. Pmid:27007866. 28.Kulkarni S, Jadhav S, Khopkar P, Sane S, Londhe R, Chimanpure V, et al. GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India.
BMC Infect Dis. Pmid:28732472. 29.Moyo S, Mohammed T, Wirth KE, Prague M, Bennett K, Holme MP, et al.
Point-of-Care Cepheid Xpert HIV-1 Viral Load Test in Rural African Communities Is Feasible and Reliable. J Clin Microbiol. Pmid:27733636. 30.Jordan JA. Evaluation of the Xpert ®HIV-1Qual Assay and Xpert ®HIV-1Quant Assay 2015. 31.WHO. WHO Prequalification of In Vitro Diagnostics.
Public Report 2017. 32.Zibusiso Ndlovu EF, Elton Mbofana,Tatenda Maparo, Daniela Garone, Carol Metcalf, Helen Bygrave, Kekeletso Kao, Sekesai Zinyowera. Multidisease testing for HIV and TB using the GeneXpert platform: A feasibility study in rural Zimbabwe. Epub 2018 Mar 2.
Q: How do I contact Technical Support? RegionTelephoneEmailUS+ 1 888 838 3222This email address is being protected from spambots. You need JavaScript enabled to view it.Australia and New Zealand+ 1800 130 821+ 0800 001 028This email address is being protected from spambots. You need JavaScript enabled to view it.Brazil and Latin America+ 8373This email address is being protected from spambots. You need JavaScript enabled to view it.China+86 400 821 0728This email address is being protected from spambots. You need JavaScript enabled to view it.France+ 33 563 825 319This email address is being protected from spambots.
Genexpert With Laptop Picture
You need JavaScript enabled to view it.Germany+ 49 69 710 480 480This email address is being protected from spambots. You need JavaScript enabled to view it.Hong Kong, Macau, Taiwan+ 8898This email address is being protected from spambots. You need JavaScript enabled to view it.India, Bangladesh, Bhutan, Nepal, and Sri Lanka+ 3010This email address is being protected from spambots. You need JavaScript enabled to view it.Italy+ 39 800 902 567This email address is being protected from spambots. You need JavaScript enabled to view it.South Africa+ 27 861 22 76 35This email address is being protected from spambots. You need JavaScript enabled to view it.United Kingdom+ 44 3303 332 533This email address is being protected from spambots. You need JavaScript enabled to view it.Other European, Middle East,and African countries+ 33 563 825 319+ 971 4 253 3218This email address is being protected from spambots.
You need JavaScript enabled to view it.Other countries not listed+ 1 408 400 8495This email address is being protected from spambots. You need JavaScript enabled to view it.Q: What information do I need to provide when contacting technical support?A: Please provide:. Name.
Institution. Contact Information. Instrument Serial NumberQ: Where do I find the serial number?A: The serial number can be found in several places.
These include the back of the instrument where the power cord is attached and at the top of the run report. Q: What are the required materials to perform Real-Time PCR?A: SmartCycler ® & software, computer configured for SC, PCR tubes, primers, probe or intercalating dye, dNTP's, MgCl2, DNA or cDNA, polymerase, water, centrifuge, pipet and pipet tips, buffers. SmartMix and OmniMix are available for convenience which contain all the reagents to perform a PCR reaction, except the primers and probes, in a SmartBead format. Q: How do I design primers and probes?A: Information regarding primer and probe design may be found in SmartNote 6.1 Designing Real-Time Assays on the SmartCycler ®II System. Q: How do I optimize my PCR reaction for Real-Time PCR on the SmartCycler®System?A: Information regarding the design of Real-Time PCR assays may be found in SmartNote 6.1 Designing Real-Time Assays on the SmartCycler ®II System.Information regarding optimizing and analyzing Real-Time Assays on the SmartCycler ® System may be found in SmartNote 6.2 Optimizing and Analyzing Real-Time Assays on the SmartCycler ® II System. Q: Which fluorophores should I label my probes with?A: Information regarding fluorophores may be found in SmartNote 6.3 Dye-Quencher Considerations for the SmartCycler ® II System.
Q: Can I run the product on a gel?A: Yes, you can run the product on a gel by simply using a gel loading pipette tip to remove the contents from the SmartCycler ® reaction tube. Q: Why are there bands of larger DNA than the PCR product on my gels?A: Possible cancatamers, which is when DNA self anneal to one another and start extending.
Q: Is it possible to perform a multiplex assay in the SmartCycler ® II system?A: Yes, the SmartCycler ® II System contains the capability to detect up to 4 targets within the same tube. More targets may be detectable by using the Advance to Next Stage feature in the software. Please refer to SmartNote Advance to Next Stage. Q: I have a positive growth curve in my negative control when I use an intercalating dye.
What is this?A: This may be a result of non-specific amplification. Please refer to SmartNote 6.1 Designing Real-Time Assays on the SmartCycler ® II System.This may be a result of primer-dimer generation. Q: When I use a protocol that contains a 30∞C hold, the processing block turns off a few minutes after the run is started.A: The acceptable temperature range is 40∞C-98∞C. If the specified temperature is not reached in 5 minutes the site will automatically stop.
The lowest temperature in the protocol must be above the internal temperature of the instrument. Q: Can I add more cycle repeats while an experiment is in progress?A: Although the software does not allow for the addition of cycle repeats while a run is progress, it is possible to program an excess of cycle repeats and stop the run or sites when appropriate. Q: Can I read optics during multiple steps in a single stage?A: No, only one optical read per stage can be programmed.
Q: Can I use the SmartCycler ® II System as a tradition PCR instrument?A: Yes, you can use the SmartCycler ® II System as a traditional thermocycler by turning the optics OFF during the cycling protocol. Q: Can I perform a melt from a high temperature to a low temperature?A: Yes. The lowest temperature must be greater than internal temperature of the instrument. Q: How many first derivative peaks are displayed on the melt graph?A: Multiple first derivative peaks indicate that multiple products were amplified during the PCR. Generally, primer-dimers melt before specific products. To change the number of melt peaks that are displayed on the graph or to change the qualifying height for the first derivative peaks, click Setup menuSystem DefaultsMelt Settings.
If the melt curve appears noisy (jagged), try smoothing the melt curve by averaging 3-5 points. Click the Setup menuSystem DefaultsMelt Settings. Q: What dyes can be used on the SmartCycler ® II System?A: The SmartCycler ® II System is calibrated for FAM, Cy3 or TET or Alexafluor532, Texas Red and Cy5 or Alexafluor647. Q: Can I use VIC, HEX or JOE on the SmartCycler ®?A: VIC, HEX, and JOE are very similar to TET and therefore may work with our calibration, but we cannot guarantee their performance since the base it is attached to may shift the optical spectrum.
To use a dye that has not been calibrated at Cepheid, we suggest running as a simplex reaction and viewing the fluorescence reading in the adjacent channels to determine any cross-talk. If the desired result is not generated, the SmartCycler ® and SmartCycler ® II system may be recalibrated with the user-defined Optical Calibration feature in the SmartCycler ® Software. Q: I created a FAM graph but it is displaying temperature.A: This is most likely due to the incorrect graph type. Check the graph definition in the Define Graphs screen to verify that the graph type is optics and not set to temperature. Q: I created a new graph but it is not listed in the View Results screen.A: When a new graph is defined in the Define Graphs screen it is saved and stored in the database but not associated with a run unless the 'Automatically add to new runs' box is checked. To view a new graph, go to the View Results screen and click the Select Graph button at the bottom of the screen.
Highlight the graph name in the 'All Graphs' column, click the right-pointing arrow then click OK. Q: When I use the print icon, the graph print out does not contain enough information, is there an alternative way to capture a picture of the data?A: To capture the entire screen use the Print screen button on your keyboard.
Simultaneously hold down the Print Screen and Alt buttons on the keyboard then open Word or Power Point and select paste.Also, it is possible to save the screen as a jpeg file and to save graphs as jpeg files. To save the screen as a jpeg file go to the Tools menu and select Save Screen to File. To save a graph as a jpeg file, right-click on the graph and select Save to file (jpg).
Q: I can't zoom on a graph when a run is in progress.A: Since data is constantly being collected and reported in real time, it is not possible to hold a zoom from a previous time point. This occurs even when viewing a saved run if another run is in progress. Q: How can I get negative fluorescence?A: This is caused by the background subtraction calculation. This usually occurs when the threshold is set too high. If the primary signal or 2nd derivative does not cross the threshold, the background subtraction calculation will continue until the end of the run.
Try decreasing the threshold value or adjusting the background min and max values. Q: Do I need to select graphs before I start a run?A: It is not necessary to define or select graphs before the run is started.
Graphs can be created before, during, or after the run is started. Q: Can I look at other runs while the instrument is in use?A: Yes, it is possible to view and analyze previous runs while the instrument is in use. Q: What is a Ct?A: The most basic definition of a Ct is the first cycle in which there is a significant increase in fluorescence above the background. Q: What is the difference between 2nd derivative and primary curve?A: The 2nd derivative and primary curve are two ways to determine a cycle threshold value.If the primary curve is used the cycle threshold reported is the intersection of the growth curve and the threshold.If the 2nd derivative is selected the software determines the cycle threshold value by calculating the 2nd derivative of the growth curve. The software chooses the largest 2nd derivative peak for the cycle threshold (this is usually the inflection point of the growth curve).
Q: What is the difference between automatic and manual Threshold Settings?A: If manual is selected, the user sets the fluorescence value used for the threshold. Manual mode gives the user more flexibility to evaluate background.Automatic will always set threshold above background, however, it does not allow the user to choose the fluorescence value used for the threshold. Automatic mode calculates the threshold as the specified number of SDs above the background. Q: I made changes to the analysis table but the graph did not update to reflect changes.A: The Update Analysis button located at the bottom of the View Results screen must be selected to implement changes made to the Analysis Settings or Results Table. Q: Is there a way to reduce the noise in my growth curves?A: The boxcar average analysis setting averages data points, which reduces the noise displayed in the growth curve. The number of points (usually 2 or 3) is specified by the user.
However, boxcar averaging can shift cycle threshold values by approximately one cycle. Q: When background subtraction is 'ON' I see drift of the baseline (positive or negative). Can this be corrected?A: The background min and max cycle numbers can be adjusted to reduce the baseline drift. Q: My negative samples are reporting Ct values in the Results Table.A: If the threshold is set too low and baseline drift crosses the threshold a Ct may be reported. Increase the threshold value (manual mode) or the number of SDs used to calculate the automatic threshold.
Q: Can I make changes to the Analysis or Results table while a run is in progress?A: Yes, changes can be made to the analysis settings at any time. Always click the Update Analysis button to implement changes made to the Results Table or Analysis Settings.Note: If the Advance to Next Stage feature is used in software version 2.0, the analysis settings must be set up before the run has started. Q: Can I use one standard curve for several runs?A: Yes, a previous standard curve can be imported and saved with a run that contains only unknown samples. Click the Import Std Curve button at the bottom of the View Results screen to select a run from a list of all the valid standard curves in the database. (Chapter 5) Q: Do I need to set-up the analysis settings before a run is started?A: No, the analysis settings can be set up before, during or after a run. Each time a change is made to the analysis settings the Update Analysis button must be selected to implement the changes.
To save changes made to the Analysis settings click the Save Run button. Q: I selected sites as standards but my standard curve graph does not show any data.A: The Update Analysis button must be selected after changes are made to the Results Table. Check that the standard values were entered in the correct column. Q: Where is the exported data saved?A: Exported data is saved to the export folder that is located in the C:DriveSmartCycler ® Export (creating a shortcut to this folder is recommended). Q: How do I see errors that have occurred?A: Notification of an error is displayed in the Check Status screen and the Results Table. To view errors that have occurred since software application was last opened go to the messages field in the Maintenance screen.To view errors that occurred before software was last opened go to the Errors folder that is located in the C: driveSmartCycler ®Errors folder. It is necessary to open notepad to view error messages.
These will also be posted on the last page of the report. Q: How do I export? What can I do with the exported data?A: Select the Export button at the bottom of the View Results screen. Select the type of data to export. Enter a file name and click Save. Q: What is the GeneXpert ® System?A: The GeneXpert Dx System automates and integrates sample preparation, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time Polymerase Chain Reaction (PCR).
The system is suited for in vitro diagnostic and research based applications that require hands-off processing of patient samples (specimens) and provides both summarized and detailed test results data in tabular and graphic formats.The GeneXpert system can only be used with the GeneXpert cartridge. Q: What is the cartridge?A: Disposable, single—use GeneXpert Dx cartridge holds the samples and reagents that you want to process in the GeneXpert Dx System. Each cartridge consists of the following components:Processing chambers—Hold samples, reagents, processed sample, and waste solutions. One chamber is designated as an air chamber to equilibrate pressures within the cartridge.Valve body-Rotates and allows fluid to move to different cartridge chambers and to the reaction tube. Within the valve body, the specimen is isolated, PCR inhibitors are removed, and specimens are ultrasonically lysed (if applicable).
After the sample is processed, it is mixed with PCR reagents and moved into the integrated reaction tube.Reaction tube—Enables rapid thermal cycling and optical excitation and detection of the tube contents. The reaction tube is automatically inserted into the I-CORE module after the cartridge is loaded into the instrument.The GeneXpert cartridges are not supplied with the Cepheid. Contact Cepheid Customer Service to order the assay specific cartridges: 1-888-838-3222 Option 1.
Q: What dyes can be used on the GeneXpert DX system?A: The GeneXpert (Diagnostic System) is calibrated with FAM, Alexa Fluor532, Texas Red, and Alexa Fluor647. Q: What dyes can be used on the GeneXpert Biothreat system?A: The GeneXpert (Biothreat System) is calibrated with FAM, VIC, DRox and LIZ Q: What assays can I run on the GeneXpert DX System?A: The GeneXpert ® Dx system can run a number of assays developed by Cepheid. Please refer to the products section of our website.
Q: What symbology is supported with the 2D scanner?A: Codes 39 and 128 are supported. Q: Can I connect a different scanner or change the configuration?A: No, we can only support the components that are shipped with the system. Do not replace or reconfigure any of the components. Using non-approved components can produce invalid results, cause loss of data, or damage other system components.
Q: How do I archive a test?A: Click on Data Management/ Archive Test and click on the test(s) you wish to archive. Click Archive and follow the defaults. When emailing an archive run, do not use a Yahoo account or a MAC computer.
Also, some filters may remove the.gxx extension. If this happens, change it to anything but.txt.
Q: How many GX instruments can be hooked up to a computer system?A: The computer can support 1 GX instrument. Agreement and Release for Unsolicited Submission.I represent that I have the authority to disclose the information in the attached Submission, and the authority to execute this Agreement and Release.I also represent that the information disclosed in the submission is wholly original to me, that I am the sole owner of all the information disclosed in the Submission, and that no interest in the information in the Submission has been granted to or acquired by others.I acknowledge and agree that Cepheid has not solicited the Submission, and that I make it voluntarily. I agree that no confidential relationship is established or implied by Cepheid’s receipt and evaluation of the Submission, that Cepheid shall have no obligation to keep any information in the Submission confidential, and that Cepheid shall have no obligation to return the Submission to me.I agree that Cepheid will have full and sole discretion with respect to what (if any) action Cepheid takes with respect to my Submission.