2019-nCOV Real-Time PCR Kit Analytical Sensitivity Study For research use only ner] \/ A1-15259 50 tests Document No: A1-18259EN_v1.0A.l. Analytical Sensitivity Study sensitivity or detection limit for nucle ¢ acid-based assays shall be expressed by the 95% positive cutoff value. This is the analyte concentration where 95% of test runs ve rial diluti n international alyzed by us (TPC) samples. The 1s Usi positive results followin; analytical sensitivity was a MATERIAL! HE 2221 Vine PCR Kit (Cat#: 09065050, 09065100 Lot No: 20-00200, 20-00227, 20-00137, 20-001 91, 20-00201, 20-00139 manufacturing date 10.2019) * RTA Viral Nucleic Acid Isolation Kit (Cat # 09029100) ER (2:21 Positive Control (TPC) # BIO-RAD CFX96-IVD Real-Time PCR Detection System (Cat. No:1855095-IVD) * Hard-Shell Thin-Wall 96-Well Skirted PCR Plates (BIO-RAD, Cat*: HSP-9655) © Microseal 'B’ Adhesive Seals. optically clear (BIO-RAD, Cat#: MSB-L001) * Disposable powder-free gloves * Micropipettes (0.5 wl — 1000 ul) Sterile micropipette tips with filters ME Three different expe ned for each gene detection mix. Experiments were done by usc of (MMM Tarset Positive Co 22 of 300, 100, 30, and 10 Copies trol (TPC). A dilutis tive Control (TPC) was prepared lL give the final concentrations I Nucleic Acid ons were extracted by RTA V Iso! to RTA Viral Nucleic Aci ion Kit accordin Iso volumes were 250 ul and elution volumes were 30 ul Then, PCR. BEE 02 ot Peo: 2 Real Time PCR Kit Handbook. Each dilution was tested in 24 replicates. BIO-RAD CFX96- actions were setup byIVD Real: PCR Detection System was used for amplification, detection and Probit analysis was done by XLSTAT statistics program. Date of the Experiments a. HR 2! y tical Sens 120 ical Sensi 14,03. 14.03.2020 14.03.2020 RESULTS. Amplification plots and Ct values of all experiments are given in the figures and tables below Lower limit was calculated by probit a1 is done by IBM SPSS statistics program. Data used by statisties program for probit analysis are shown in Tables 3 and 3. Probit analysis was mix, Limit of Detection (LoD) values and 95, attached at the end of each section. For each g % confidence ra are summarized in Table 1. a: confidence ranges ‘Table 1 ime PCR Kit - Limit of Detection (Lo! ) values and 95 E 38 33 50 RdRP 38 31 38 HR Bea Fime PCR Kit Analytical Sensitivity Studies for target gene E: Ct valuesAmplification Figure 1: Ral Time PCR Kit Analytical Sensitivity Study | — Amplification Plots Ampifiestien a0 100 2 B 100 =o 3 0 20 0 “ cycles Figure 2: EE Real Time PCR Kit Analytical Sensitivity Study 2 — Amplification Plots Amplification Figure 3: EE Real Time PCR Kit Analytical Sensitivity Study 3 — Amplification Plots‘Table 3: IIR cal Time PCR Kit Analytical Sensitiv Probit Analysis for target gene E y Study Data used by XLSTAT for Concentration of | Replicate # of’ | Positive # of Sample Samples Samples (COPIES/ml) 300 m4 24 100 Fa 24 30 M 18 10 4 1 Probit Analysis of SARS-CoV-2 Real Time PCR Kit for Target Gene E PROBIT VARO0003 OF VARO0002 WITH VAROO00L LOG NONE MODEL PROBIT PRINT FREQ C1 (CRITERIA P(0.15) ITERATE(20) STEPLIMIT(.11. Probit Analysis Notes Output Created es a Active Dataset Fiter Weight Spi Nof Rows in Woeking Data File Missing Value Handling Definition of Missing Cases Used Syntax Resources 20-MAR-2020 09:57:40 DataSet User-defined missing values are treated! as missing. Statistics are based on all eases with valed data far all variables ‘im the mexdel. PROBIT VARUOOD3 OF VAROO0O2 WITH WA ROQO0L LOG NONE MODEL PROBIT PRINT FREQ C1 CRITERIA PULLS) ITERATE(20) STEPLIMITi.1). 0-00-00,39 tapsed Time 5.Data Information Nof Cases Naka 4 Rejected Missing o Number of Responses o Number of Subjects Control Group o Convergence Information Numbe Optimal Solution Herations, Found PROBIT 19_Yes Parameter Estimates 95% Confidence Interval Parameter ic Std. Error a Lower Bound PROBIT, —_VAROOOOL 120 27 4491 00K 68 Intercept 22.935, TL 41st oo 23.636. Parameter Estimates PROBIT. model: PROBIT pl ~ Intercept + BX ‘Chi-Square Tests Chi-Square df PROBIT Pearson Goodiness-of-Fit Test baw 2 1000, a. Since the significance level ts greater than ,150, no beterogencity Factor is wsed in the calculation of confidence lim b. Statistics based om individual cases differ from statistics bused on aggregated cases.Cell Counts and Residuals, Number af Observed Number VAROODL Subjects PROBIT 1 300,000, mM 000. 2 100,000 uM 00 3 0.0000 w 18.000 000 4 10.000, L Loan, 000, ‘Cell Counts and Residuals Number PROBIT 1 1.000, i Probubuley PROBIT 010 020 030) an “2688 isis aso “11st 16,090 060) 190 16668 70. L201 aTa78 se 2.192 17639 30) 3490 18062 100 ROM 1kass 150 7.283 20,119 200) 9.901 21,502 250) 12490 274s 300 13.997 23.99 350 1s.704 25,069 400 17,259 26.204 450 18,695 27410 500 304 04087 28.649 sso 25.438 21,305 29.961 40 26.499 21 31.368 ssn 27.596. 23.703 32,05 700 28.382 24.478 34,597 750 30,000 26075 36.462 0 31380. S80) 33,008 28737Confidence Limits 98% Confidence Limits for VAROOOOL Probably Estimate Lower Bound peered s10 35.538 30,813 45,406 920 36.073 31238 930 36.660 31,700 340 32.317, 216 32,796 33,472 51,165 3204 sa014 41464 35375 55485 43.730 37.058 59,400 = Probit Transformed Reeponses Probit ast om 708 m0 \VAR00001 HR 21 tine PCR Kit Analytical Sensitivity Studies for Target Gene RAR Table 4: MEE Real Time PCR Kit Analytical Sensitivity Studies for Target Gene RARP — Ct values 3138 | 43,49Apliicstion = & cycles wvity Study | — Amplification Plots for Target Gene RARP HE s21stical sc Amplification 3000 Sens 2800 2000 B iso 1000 500 ° 19 20 » 2 Cycles sitivity Study 2- Amplification Plots for Tangct Gene RARP Ampltestion nu 8 8 Cyc HE *2!y'1cal Sensitivity Study 3— Amplification Plots for Target Gene RARP -Table 5: | Time PCR Kit Analytical Sensiti c RRP Study Data used by SPSS for Probit Concentration of | Replicate # of | Positive # of Samples Samples (COPIES/ml) 300 4 24 100) 2a u 30 24 20 10 4 6 Probit Analysis of BB Real Time PCR Kit for Target Gene RURP PROBIT VAR00003 OF VAR00002 WITH VAROOOOL LOG NONE MODEL PROBIT PRINT FREQ C ‘CRITERIA P(0_15) ITERATE(20) STEPLIMIT(. 1). Probit Analysis Notes napa Crete 2o.MAR-2070 10:2 ca = Active Date Datsett Fier one Wei one Split Fite one N of Roms ia Working Data ‘ File ising Value Handi eee Kia crcefinad msing wok ora tented ws ici Cases Used Sttstics are based om all cases vith ali da fr all variables inthe model Snax PROBIT VARUIOS OF ‘VaRo002 WITH VARUOROL LOG NONE MODEL PROBIT PRINT FREQ C1 CRITERIA PAS) :RATE(20) STEPLIMITL). ia Pcie Teas 0.00 00.2 ed Time 0-00-0020)Data Information of Cases Nalid 4 Rejected Missing L Number of Responses o Number of Subjects ‘Control Group o Convergence Information Number of Optimal Solution erations Found PROBIT 17 Yes Parameter Estimates 99% Confidence val Parameter Estimate Sid. Error z Siz Lower Bound PROBITs —_VAROODOL 082 21 3.981 00 Oa Intercept 1495 Aa 3.367 0 1,930 Parameter Estimates 95% Confidence Interval Parameter Upper Bound PROBITS VARDOOOL 12: Intercept L051 a. PROBIT model: PROBIT(p) = Intercept + BX Chi-Square Tests chisgue | a Sig PROBIT Pearson Goodness Tew. 0000 2 1000. a. Since the significance level is greater than ,150, no beteragencity factor is used in the calculation of confidence limits. bb. Statistics based on individual cases differ from statistics based on aggregated cases,Cell Counts and Residuals Observed Expected her VARINODL Subjects Responses Responses Residual PRoBIT 1 300,000 2m x 24,000 bo 2 100.000 24 Fa 24,000 000 3 30,000 x 20 20.000 0a 4 40.000 a 6 6,000 0 Cell Counts and Residuals Number Probability PROBIT L 1.000 2 1.000 3 aaa 4 250 Confidence Limits .e Limit AR OOO robb Estimate Upper Hound PROBIT 10 10,121 336 20 4.801 2661 30 4.504 4149 040 Sz 050 6.202 060 6M 70 2.093. ak 8324 90 KOOL 100) 936 150 11.699) 200 13.571 250 15.258 0 16,854 350 13.522 S082 1136 20 15.130 7.866 20.085 450 16.685 10,067 21,760 S00 In216 12.202 23,595 530. 19,747 14,306, eat 21.302 16.195 580 2.900 1976Confidence Limits 98% Confidence Limits for VARO0001 Probability Estimate Lower Bound Upper Bound 300 ae 19,692 3300 0? tatn) ss 2h 350 40841 2513 +4080 may 49,642 0 54 7g 51000 ss 330 34,192, 2207 4114 EIAs) 018 3M 718 039 Oss as 970 41126 J i2 a0 43232 34200 b1614 3 f28 O42 Probit Transformed Responses ia a3 i 0 4 * aa ‘ant 1808 te WAROO001