Interface design recommendations for computerised clinical audit and feedback: Hybrid usability evidence from a research-led system.

Interface design recommendations for computerised clinical audit and feedback: Hybrid usability evidence from a research-led system.
Audit and Suggestions (A&F) is a extensively used high quality enchancment method that measures clinicians’ medical efficiency and reviews it again to them. Computerised A&F (e-A&F) system interfaces could consist of 4 key parts: (1) Summaries of medical efficiency; (2) Affected person lists; (3) Affected person-level knowledge; (4) Beneficial actions.
There’s a lack of proof relating to how you can greatest design e-A&F interfaces; establishing such proof is vital to maximising usability, and in flip enhancing affected person security.
To judge the usability of a novel theoretically-informed and research-led e-A&F system for major care (the Efficiency Enchancment plaN GeneratoR: PINGR).
(1) Describe PINGR’s design, rationale and theoretical foundation; (2) Establish usability points with PINGR; (3) Perceive how these points could intervene with the cognitive targets of end-users; (4) Translate the problems into suggestions for the user-centred design of e-A&F methods.
Eight skilled well being system evaluators carried out a usability inspection utilizing an modern hybrid method consisting of 5 phases: (1) Improvement of consultant consumer duties, Objectives, and Actions; (2) Combining Heuristic Analysis and Cognitive Walkthrough strategies right into a single protocol to establish usability points; (3) Consolidation of points; (4) Severity ranking of consolidated points; (5) Evaluation of points in keeping with usability heuristics, interface parts, and Objective-Motion construction.
A remaining checklist of 47 points have been categorised into eight heuristic themes. Essentially the most error-prone heuristics have been ‘Consistency and requirements’ (13 usability points; 28% of the overall) and ‘Match between system and actual world’ (n=10, 21%).
The really helpful actions part of the PINGR interface had probably the most usability points (n=21, 45%), adopted by patient-level knowledge (n=5, 11%), affected person lists (n=4, 9%), and summaries of medical efficiency (n=4, 9%). Essentially the most error-prone Actions throughout all consumer Objectives have been: (1) Affected person choice from a listing; (2) Information identification from a determine (each population-level and patient-level); (3) Disagreement with a system advice.
By contextualising our findings throughout the wider literature on well being info system usability, we offer suggestions for the design of e-A&F system interfaces regarding their 4 key parts, along with how they could be built-in inside a system.
Interface design suggestions for computerised medical audit and suggestions: Hybrid usability proof from a research-led system.

It All Simply Clicks: Improvement of an Inpatient E-Seek the advice of Program.

Though the usage of digital consultations (e-consults) within the outpatient setting is commonplace, there may be little proof of their use within the inpatient setting. Usually, the one selection hospitalists have is between requesting a time-consuming in-person session or requesting an off-the-cuff, undocumented “curbside” session. For a brand new, distant hospital in our healthcare system, we developed an e-consult protocol that can be utilized to handle easy session questions.

Within the first yr of this system, 143 e-consults occurred; the highest 5 consultants have been infectious illness, hematology, endocrinology, nephrology, and cardiology. Over the primary Four months, no questions of safety have been recognized in chart overview audits; thus far, no questions of safety have been recognized by the hospital’s incident reporting system. In surveys, hospitalists have been universally happy with the standard of e-consult suggestions, although solely 43% of consultantsagreed.

With acceptable take care of affected person choice, e-consults can be utilized to securely and effectively present subspecialty experience to a distant inpatient web site Journal of Hospital Medication 2017;12:332-334.

Rapid sequence induction (RSI) in trauma patients: Insights from healthcare providers.

Rapid sequence induction (RSI) in trauma patients: Insights from healthcare providers.

We aimed to explain the present apply of emergency physicians and anaesthesiologists within the choice of medication for rapid-sequence induction (RSI) amongst trauma sufferers.A potential survey audit was performed based mostly on a self-administered questionnaire amongst two intubating specialties.

The popular kind and dose of hypnotics, opioids, and muscle relaxants used for RSI in trauma sufferers had been sought within the questionnaire. Information had been in contrast for using induction agent, opioid use and muscle relaxant amongst secure and unstable trauma sufferers by the intubating specialties.

RESULTS

A complete of 102 individuals had been included; 47 had been anaesthetists and 55 had been emergency physicians. Propofol (74.5%) and Etomidate (50.0%) had been probably the most continuously used induction brokers. Considerably increased proportion of anesthesiologist used Propofol whereas, Etomidate was generally utilized by emergency physicians in secure sufferers (P=0.001).

mergency physicians most popular Etomidate (63.6%) and Ketamine (20.0%) in unstable sufferers. The 2 teams had been comparable for opioid use for secure sufferers. In unstable sufferers, use of opioid differed considerably by intubating specialties.

The relation between rocuronium and suxamethonium use did change among the many anaesthetists. Emergency physicians used extra suxamethonium (55.6% vs. 27.7%, P=0.01) in secure in addition to unstable (43.4 % vs. 27.7%, P=0.08) sufferers.There’s variability in using medication for RSI in trauma sufferers amongst emergency physicians and anaesthesiologists. There’s a have to develop an RSI protocol utilizing standardized sorts and dose of those brokers to ship an efficient airway administration for trauma sufferers.

Speedy sequence induction (RSI) in trauma sufferers: Insights from healthcare suppliers.

‘Screening audit’ as a high quality assurance device in good scientific apply compliant analysis environments.

With the rising quantity of scientific analysis, laws and analysis ethics have gotten extra stringent. This development introduces a necessity for high quality assurance measures for guaranteeing adherence to analysis ethics and human analysis safety past Institutional Evaluation Board approval.
 Audits, probably the most efficient instruments for assessing high quality assurance, are measures used to judge Good Medical Observe (GCP) and protocol compliance in scientific analysis. Nevertheless, they’re laborious, time consuming, and require experience.
Due to this fact, we developed a easy auditing course of (a screening audit) and evaluated its feasibility and effectiveness.
The screening audit was developed utilizing a routine audit guidelines based mostly on the Severance Hospital’s Human Analysis Safety Program insurance policies and procedures. The measure contains 20 questions, and outcomes are summarized in 5 classes of audit findings. We analyzed 462 research that had been reviewed by the Severance Hospital Human Analysis Safety Middle between 2013 and 2017.
We retrospectively analyzed analysis traits, reply charge, audit findings, related components and post-screening audit compliance, and so forth. RESULTS: Investigator reply charges progressively elevated, apart from the primary 12 months (73% → 26% → 53% → 49% → 55%). The research had been graded as “important,” “main,” “minor,” and “not a discovering” (11.9, 39.0, 42.9, and 6.3%, respectively), based mostly on findings and variety of deficiencies. The auditors’ selections confirmed honest settlement with weighted kappa values of 0.316, 0.339, and 0.373. Low-risk degree research, single heart research, and non-phase scientific analysis confirmed extra prevalent frequencies of being “main” or “important” (p = 0.002, < 0.0001, < 0.0001, respectively).
Inappropriateness of paperwork, failure to acquire knowledgeable consent, inappropriateness of knowledgeable consent course of, and failure to guard individuals’ private data had been related to increased audit grade (p < 0.0001, p = 0.0001, p < 0.0001, p = 0.003).
We had been in a position to observe important GCP violations within the routine inside audit outcomes of post-screening audit compliance checks in “non-responding” and “important” research upon making use of the screening audit.
Our screening audit is an easy and efficient strategy to assess total GCP compliance by establishments and to make sure medical ethics. The device additionally supplies helpful choice standards for conducting routine audits.

Types and Frequency of Infusion Pump Alarms and Infusion-Interruption to Infusion-Recovery Times for Critical Short Half-Life Infusions: Retrospective Data Analysis.

Types and Frequency of Infusion Pump Alarms and Infusion-Interruption to Infusion-Recovery Times for Critical Short Half-Life Infusions: Retrospective Data Analysis.

Alarm fatigue generally results in a diminished response to alarms. Acceptable and well timed response to intravenous pump alarms is essential to infusion continuity.

The problem of filtering out vital quick half-life infusion alarms from nonurgent alarms is a key problem for danger administration for clinicians.

Crucial care areas present ample alternatives for intravenous treatment error with the frequent administration of high-alert, vital quick half-life infusions that require rigorous upkeep for continuity of supply.

Most critical treatment errors in vital care happen in the course of the execution of therapy, with performance-level failures outweighing rule-based or knowledge-based errors.One goal of this research was to ascertain baseline knowledge for the categories and frequency of alarms that vital care clinicians are uncovered to from quite a lot of infusion units, together with each giant quantity pumps and syringe drivers.

One other goal was to establish the quantity of those alarms that particularly relate to vital quick half-life infusions and to judge consumer response occasions to alarms from infusion units delivering these explicit infusions.The occasion logs of 1183 infusion pumps utilized in vital care environments and generally care areas throughout the European area had been mined for a variety of alarm states.

The research then centered on a choice of infusion alarms from units delivering vital quick half-life infusions that will warrant speedy consideration from clinicians in an effort to keep away from probably dangerous extended infusion interruption.

Types and Frequency of Infusion Pump Alarms and Infusion-Interruption to Infusion-Recovery Times for Critical Short Half-Life Infusions: Retrospective Data Analysis.
Sorts and Frequency of Infusion Pump Alarms and Infusion-Interruption to Infusion-Restoration Occasions for Crucial Quick Half-Life Infusions: Retrospective Information Evaluation.

The response time of clinicians to infusion-interruption states and alarms for the chosen vital quick half-life infusions was then calculated.

Preliminary evaluation confirmed a imply common of 4.50 alarms per infusion within the basic vital care pump inhabitants versus the entire hospital fee of 1.39. Within the pediatric intensive care unit (PICU) group, the alarms per infusion worth was considerably above the imply common for all vital care areas, with 8.61 alarms per infusion. Infusion-interruption of vital quick half-life infusions was discovered to be a major drawback in all areas of the final vital care pump inhabitants, with a major variety of downstream (ie, vein and entry) occlusion occasions famous.

Whereas the imply and median response occasions to vital quick half-life infusion interruptions had been typically throughout the half-lives of the chosen medicines, there was a excessive prevalence of outliers by way of response occasions for all of the vital quick half-life infusions studied.

This research offers a sign of what may be anticipated in vital care environments by way of the quantity of basic infusion alarms and significant quick half-life infusion alarms, in addition to for clinician response occasions to vital quick half-life infusion-interruption occasions.

This research additionally identifies probably problematic areas of the hospital for alarm fatigue and for explicit problems with infusion and infusion-line administration.

Utility of the proposed protocols will help create benchmarks for pump alarm administration and clinician response occasions. These protocols might be utilized to research on the affect of alarm fatigue and for the analysis of protocols, infusion-monitoring methods, and infusion pump-based treatment security software program aimed toward decreasing alarm fatigue and guaranteeing the upkeep of vital quick half-life infusions. Given the frequency of infusion alarms seen on this research, the chance of alarm fatigue because of the white noise of pump alarms current in vital care, to which clinicians are continuously uncovered, may be very excessive.

Moreover, the added difficulties of sustaining vital quick half-life infusions, and different infusions in specialist areas, are made clear by the excessive ratio of downstream occlusion to infusion begins within the neonatal intensive care unit (NICU).

The power to quantitatively observe the quantity of alarms and clinician response occasions contributes to a better understanding of the problems of alarm fatigue in intensive care models. This may be utilized to medical audit, can permit for focused coaching to cut back nuisance alarms, and might assist in planning for enchancment in the important thing space of upkeep of steady-state plasma ranges of vital quick half-life infusions. One clear conclusion is that the treatment administration rights needs to be prolonged to incorporate proper upkeep and ensured supply continuity of vital quick half-life infusions.

Coronavirus (2019-nCoV) Real Time RT-PCR Kit(Cat. #: RR-0478-02)

2019-ncorv qPCR
Wuhan, China and a new coronavirus (CoV) has been identified as a possible pathogen causing this outbreak.

 

Please be advised that we have successfully developed Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit(Cat. #: RR-0478-02) to detect this new coronavirus.

1250 Euro from Gentaur

Some countries have stepped up preventive measures including airport temperature screening for travelers arriving from Wuhan in the wake of this outbreak.

nCorV NatTrol nucleic acid control can be used as positive control for this qPCR test.

Coronavirus (2019-nCoV) 

2019-ncorv qPCR
primers for ncorv

For use with

  • ABI Prism®7500/7900;
  • Bio-Rad CFX 96;
  • Rotor Gene™6000;
  • Bio-Rad CFX96
  • SLAN;MICPOCDx 48 Instrument

1. Intended Use

Novel Coronavirus (2019-nCoV) Real Time Multiplex RT-PCR Kit is used for the qualitative detection of a novel coronavirus, which was identified in 2019 at Wuhan City, Hubei Province, China, in upper respiratory tract specimens (nasopharyngeal extracts, deep cough sputum, etc.) and lower respiratory tractspecimens(alveoli irrigationfluid,etc.) by realtime PCR systems.

2. Principle of Real-TimePCR

The Gentaur principle of the real-time detection is based on the fluorogenic 5’nuclease assay. During the PCR reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR detection system. The PCR cycle at which an increase in the fluorescence signal is detected initially (Ct) is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities in real time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification.

3. Product Description

On January 11, 2020, Chinese health authorities preliminarily identified more than 40 human infections with a novel coronavirus in an outbreak of pneumonia under investigation in Wuhan City, Hubei Province, China. The Chinese authorities identified a new type of coronavirus (novel corona-virus,named as 2019-nCoV), which was isolated on7 January 2020. Corona-viruses are a large family of viruses, some causing illness in human and others circulating among animals such as camels, cats and bats. 2019-nCoV is a novel coronavirus. The primer and probe design for this kit is based on the newly released strain (2019-nCoV) (GeneBank accession: MN908947) and covers 6 2019-nCoV strains sequences (EPI_ISL_402119, EPI_ISL_402120, EPI_ISL_402121,EPI_ISL_402122,EPI_ISL_402123, EPI_ISL_402124included). The kit contains a specific ready-to-use system for the detection of Novel Coronavirus (2019-nCoV) by Reverse transcription Polymerase Chain Reaction (RT-PCR) in the real-time PCR system. The master contains a Super Mix forthe specific amplification of virus RNA. The reaction is done in one step real time RT-PCR. The first step is a reverse transcription (RT), during which the virus RNA is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used to amplify the specific gene fragments by means of polymerase chain reaction (PCR). Fluorescence is emitted and measured by the real time systems´ optical unit during PCR. The detection of amplified virus DNA fragment is performed in fluorimeter channel FAM, HEX/VIC/JOE and Cal Red 610/ ROX/TEXASRED with the fluorescent quencher BHQ1. NatTrol can be used as control.

4. Kit Contents

Ref. Typeofreagent Presentation 25rxns 1 2 3 4 NovelCoV(2019-nCoV)SuperMix RT-PCREnzymeMix NovelCoV(2019-nCoV)NegtiveControl NovelCoV(2019-nCoV)PositiveControl 1vial, 480l 1vial, 28l 1vial, 400μl 1vial, 30μl Analysissensitivity:1×103 copies/ml; Note: Analysis sensitivity depends on the sample volume, elution volume, nucleic acid extraction method and other factors. If you use the RNA extraction kits recommended, the analysis sensitivity is the same as it declares. However, when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method, it can be much higher.

5. Storage

•Allreagents shouldbestoredat-20°C.Storageat+4°Cisnotrecommended. •Allreagents canbeuseduntiltheexpirationdateindicatedonthekitlabel. • Repeated thawing and freezing (> 3x) should be avoided, as this may reduce the sensitivity of theassay. •Coolallreagentsduringtheworkingsteps. •SuperMixshouldbestoredinthedark.

6. Additionally Required Materials and Devices

•Biological cabinet •Vortexmixer •Cryo-container •Sterilefiltertipsformicro pipets •Disposable gloves,powderless •RefrigeratorandFreezer •RealtimePCRsystem •RealtimePCRreactiontubes/plates •Pipets(0.5μl–1000μl) •Sterilemicrotubes •Biohazardwastecontainer •Tuberacks •Desktopmicrocentrifuge for“eppendorf”typetubes(RCFmax.16,000xg)

7. Warnings and Precaution

Carefullyreadthisinstructionbeforestartingtheprocedure. •Thisassayneedstobecarriedoutbyskilledpersonnel. •Clinicalsamplesshouldberegarded aspotentiallyinfectious materialsand shouldbepreparedinalaminarflowhood. •ThisassayneedstoberunaccordingtoGoodLaboratoryPractice. •Donotusethekitafteritsexpirationdate. •Avoidrepeatedthawingandfreezing ofthereagents,thismayreducethesensitivityofthetest. •Oncethereagentshavebeenthawed,vortexandcentrifugebrieflythetubesbeforeuse. •PreparequicklytheReactionmixoniceorinthecoolingblock. • Set up two separate working areas: 1) Isolation of the RNA/ DNA and 2) Amplification/ detectionofamplificationproducts. •Pipets,vialsandotherworkingmaterialsshouldnotcirculateamongworkingunits. •Usealways sterilepipettetipswithfilters. •Wearseparate coatsandglovesineacharea.

Measles Virus Detection

1. Intended Use Measles virus real time RT-PCR Kit is used for the detection of measles virus in nasal and pharyngeal secretions and urine samples by using real time PCR systems. This virus can also be detected in many other kinds of samples such as blood, serum, plasma liquor, etc., but not very common. 2. Principle of Real-Time PCR The principle of the real-time detection is based on the fluorogenic 5’nuclease assay. During the PCR reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR detection system. The PCR cycle at which an increase in the fluorescence signal is detected initially (Ct) is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification. 3. Product Description Measles is one of the most contagious of all human viruses, with about forty million infections world wide each year, and one to two million deaths. Measles outbreaks are common in underdeveloped countries where there is lower socioecoomic status, crowding, and low access to health care. In the third world, there may be up to 900,000 measles related deaths per year. Therefore, there is a lot of pressure on health in different countries in controlling the disease through vaccination. Measles causes rash, cough, and fever, and can lead to ear infection, pneumonia, conjunctivitis, diarrhea, seizures, brain damage, and death. Measles virus real time RT-PCR kit contains a specific ready-to-use system for the detection of the measles virus by Reverse Transcription Polymerase Chain Reaction (RT-PCR) in the real-time PCR system. The master contains a Super Mix for the specific amplification of measles virus RNA. The reaction is done in one step real time RT-PCR. The first step is a reverse transcription (RT), during which the measles virus RNA is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used to amplify the specific gene fragments by means of polymerase chain reaction (PCR). Fluorescence is emitted and measured by the real time systems ´optical unit during the PCR. The detection of amplified measles virus DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1. In addition, the kit contains a system to identify possible PCR inhibition by measuring the HEX/VIC/JOE fluorescence of the internal control (IC). An external positive control(1×107copies/ml) contained, allows the determination of the gene load. For further information, please refer to section 9.3 Quantitation. 4. Kit Contents Ref. Type of reagent Presentation 25rxns 1 2 3 4 5 MV Super Mix RT-PCR Enzyme Mix Molecular Grade Water Internal Control (IC) MV Positive Control(1×107copies/ml) 1 vial, 480l 1 vial, 28l 1 vial, 400μl 1 vial, 30μl 1 vial, 30μl Analysis sensitivity: 1×103copies/ml; LOQ: 2×103~1×108copies/ml Note: Analysis sensitivity depends on the sample volume, elution volume, nucleic acid extraction methods and other factors .If you use the RNA extraction kits recommended, the analysis sensitivity is the same as it declares. However, when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method, it can be much. 5. Storage • All reagents should be stored at -20°C. Storage at +4°C is not recommended. • All reagents can be used until the expiration date indicated on the kit label. • Repeated thawing and freezing (> 3x) should be avoided, as this may reduce the sensitivity of the assay. • Cool all reagents during the working steps. • Super Mix should be stored in the dark. 6. Additionally Required Materials and Devices • Biological cabinet • Vortex mixer • Cryo-container • Sterile filter tips for micro pipets • Disposable gloves, powderless • Refrigerator and Freezer • Real time PCR system • Real time PCR reaction tubes/plates • Pipets (0.5μl – 1000μl) • Sterile microtubes • Biohazard waste container • Tube racks • Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g) 7. Warnings and Precaution Carefully read this instruction before starting the procedure. • For in vitro diagnostic use only. • This assay needs to be carried out by skilled personnel. • Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood. • This assay needs to be run according to Good Laboratory Practice. • Do not use the kit after its expiration date. • Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test. • Once the reagents have been thawed, vortex and centrifuge briefly the tubes before use. • Prepare quickly the Reaction mix on ice or in the cooling block. • Set up two separate working areas: 1) Isolation of the RNA/ DNA and 2) Amplification/ detection of amplification products. • Pipets, vials and other working materials should not circulate among working units. • Use always sterile pipette tips with filters. • Wear separate coats and gloves in each area. • Do not pipette by mouth. Do not eat, drink, smoke in laboratory. • Avoid aerosols

pcr
measles

8. Sample Collection, Storage and transport • Collected samples in sterile tubes; • Specimens can be extracted immediately or frozen at -20°C to -80°C. • Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9. Procedure 9.1 RNA-Extraction Different brand RNA extraction kits are available. You may use your own extraction systems or the commercial kit based on the yield. For the RNA extraction, please comply with the manufacturer’s instructions. The recommended Extraction kit is as follows: Nucleic Acid Isolation Kit Cat. Number Manufacturer RNA Isolation Kit ME-0010/ME-0012 ZJ Biotech QIAamp Viral RNA Mini extraction Kit (50) 52904 QIAGEN 9.2 Internal Control It is necessary to add internal control (IC) in the reaction mix. Internal Control (IC) allows the user to determine and control the possibility of PCR inhibition. Add the internal control (IC) 1μl/rxn and the result will be shown in the HEX/VIC/JOE. 9.3 Quantitation The kit can be used for quantitative or qualitative real-time RT-PCR. A positive control defined
as 1×10*7

PCR
Measles sample

copies/ml is supplied in the kit. For performance of quantitative real-time PCR, Standard dilutions must prepare first as follows. Molecular Grade Water is used for dilution. Dilution is not needed for qualitative real-time PCR detection.
Take positive control (1×10 7
copies/ml) as the starting high standard in the first tube. Respectively pipette 36ul Molecular Grade Water into next three tubes. Do three dilutions as the following figures:

To generate a standard curve on the real-time system, all four dilution standards should be used and defined as standard with specification of the corresponding concentrations. Attention: A. Mix thoroughly before next transfer.
B. The positive control (1×10 7
copies/ml) contains high concentration of the target DNA. Therefore, be careful during the dilution in order to avoid contamination. 9.4 RT-PCR Protocol The Master Mix volume for each reaction should be pipetted as follows:

※PCR system without HEX/VIC/JOE channel may be treated with 1μl Molecular Grade Water instead of 1μl IC. 1) The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples, which includes the number of controls, standards, and sample prepared. Molecular Grade Water is used as the negative control. For reasons of unprecise pipetting, always add an extra virtual sample. Mix completely then spin down briefly in a centrifuge. 2) Pipet 20μl Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate/tubes. Separately add 5μl RNA sample, positive and negative controls to different reaction plate/tubes. Immediately close the plate/tubes to avoid contamination. 3) Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes. 4) Perform the following protocol in the instrument: 45°C for 10min 1cycle Selection of fluorescence channels 95°C for 15min 1cycle FAM Target Nucleic Acid 95°C for 15sec, 60°C for 1min ( Fluorescence measured at 60°C) 40cycles HEX/VIC/JOE IC 5) If you use ABI Prism® system, please choose “none” as passive reference and quencher. 10. Threshold setting: just above the maximum level of molecular grade water. 11.Calibration for quantitative detection: Input each concentration of standard controls at the end of run, and a standard curve will be automatically formed. 12.Quality control: Negative control, positive control, internal control and QS curve must be performed correctly, otherwise the sample results is invalid. Channel Control Ct value FAM HEX/VIC/JOE Molecular Grade Water UNDET 25~35 Positive Control(qualitative assay) ≤35 —— QS(quantitative detection) Correlation coefficient of QS curve≤-0.98 13. Data Analysis and Interpretation The following sample results are possible: Ct value Result Analysis FAM HEX/VIC/JOE 1# UNDET 25~35 Below the detection limit or negative 2# ≤38 —— Positive; and the software displays the quantitative value 3# 38~40 25~35 Re-test; If it is still 38~40, report as 1# 4# UNDET UNDET PCR Inhibition; No diagnosis can be concluded.

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