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The evaluation of 7 commercial real-time PCR kits for Zaire ebolavirus using virus-like particle–encapsulated EBOV RNA
The evaluation of seven commercial real-time PCR kits for Zaire Ebolavirus using virus-like particles encapsulated EBOV RNA Guojing Wang, Rui Zhang, Le Chang, Dong Zhang, Lei Zhang, Xin Yang, Chao Liu, Jiansheng Ding, Kuo Zhang, Guigao Lin, Xie Jiehong, Jinming Li PII: DOI: Reference:
S0732-8893(15)00289-8 doi: 10.1016/j.diagmicrobio.2015.07.025 DMB 13885
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
14 May 2015 8 July 2015 30 July 2015
Please cite this article as: Wang Guojing, Zhang Rui, Chang Le, Zhang Dong, Zhang Lei, Yang Xin, Liu Chao, Ding Jiansheng, Zhang Kuo, Lin Guigao, Jiehong Xie, Li Jinming, The evaluation of seven commercial real-time PCR kits for Zaire Ebolavirus using viruslike particles encapsulated EBOV RNA, Diagnostic Microbiology and Infectious Disease (2015), doi: 10.1016/j.diagmicrobio.2015.07.025
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ACCEPTED MANUSCRIPT Title page The evaluation of seven commercial real-time PCR kits for Zaire
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Ebolavirus using virus-like particles encapsulated EBOV RNA Guojing Wang1, 2, Rui Zhang1, Le Chang1, 2, Dong Zhang1, 2, Lei Zhang1, 3, Xin
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Yang1, 2, Chao Liu1, 2, Jiansheng Ding1, 2, Kuo Zhang1, Guigao Lin1, Xie Jiehong1,
1
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Jinming Li1*
National Center for Clinical Laboratories, Beijing Hospital of National Health and
Family Planning Commission, Beijing, People’s Republic of China Graduate School, Peking Union Medical College, Chinese Academy of Medical
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2
Fifth School of Clinical Medicine, Peking University, Beijing, People’s Republic of
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China
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3
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Sciences, Beijing, People’s Republic of China
*Corresponding author Mailing address: National Center for Clinical Laboratories Beijing Hospital, No.1 Dahua Road, Dongdan, Beijing 100730, P R China. Tel: 86-10-58115053; Fax: 86-10-65212064 E-mail: [email protected] Running title: Evaluation of EBOV real-time RT-PCR kits Word counts of the abstract: 143 words Word counts of the body of the text: 2539 words
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ABSTRACT
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Several Chinese commercial real-time PCR kits for Zaire Ebolavirus have been developed after the Ebola outbreak and used by Chinese medical teams in West
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Africa. In order to know the essential performance indicators of these kits, analytical sensitivity and precision were evaluated with virus-like particles (VLPs)
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encapsulated EBOV RNA. The limit of detection and the precision were completed with a series of VLPs. The maximum and minimum of limit of detection was acquired by ZJ BioTech and Daan gene respectively. For precision, all of the
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detection results were less than 5% except the maximum 5.17%. Among them
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Puruikang, Daan gene, Sansure, ZJ BioTech and Huada demonstrated superior reproducibility. Overall, the requirements of LOD＜1000 copies/mL and CV＜5%
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could be satisfied by all kits except Kehua. Meanwhile, it is feasible for VLPs as a
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substitute of positive samples in assays evaluation.
Keywords: Ebola virus, kits evaluation, real time RT-PCR, virus-like particles.
ACCEPTED MANUSCRIPT INTRODUCTION Ebola virus belongs to the Filoviridae family and contains a single negative-sense,
phosphoprotein
(VP35),
matrix
protein
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nearly 19kb RNA strand which encodes seven proteins: nucleoprotein (NP), (VP40),
glycoprotein
(GP),
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replication-transcription protein (VP30), matrix protein (VP24) and RNA dependent RNA polymerase (L) (Fields BN et al., 2007). Among the five species (Bundibugyo,
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Sudan, Zaire, Tai Forest and Reston), the Zaire species has the most frequent outbreaks and the highest mortality rate from 57% to 90% (McElroy AK et al., 2014) to human being. In 2014, viral isolation and sequence alignment were conducted and
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(Baize S et al., 2014).
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identified that the epidemic strain in West Africa was Zaire Ebolavirus (EBOV)
In early August, 2014, the World Health Organization (WHO) had announced the
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outbreak of Ebola virus disease (EVD) in West Africa as “public healthy emergency
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of international concern”. The number of both infectors and deaths of EVD in recent epidemic was the highest and more than 22 prior outbreaks in total since the firstly identified in 1976 (WHO, 2014). The center for EBOV plague was Guinea, Liberia, and Sierra Leone, along with other countries in West Africa, such as Nigeria and Senegal. Europe and the United States had also been reported as sporadic affected regions (Ledgerwood JE et al., 2014). Confronting with the situation above, China authority had provided supports for Africa since September 2014. Sierra Leone - China friendship hospital was built and a mobile biosafety level-3 laboratory (BSL-3), diagnostic reagents for EBOV, public
ACCEPTED MANUSCRIPT health experts and medical staffers were all transported and dispatched for four times to EBOV-affected areas (Gao GF et al., 2014). In response to the aid of West Africa,
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plenty of commercial kits for qualitative detecting Zaire Ebola virus had been independently developed in China.. The ZJ BioTech kit had obtained the fully
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approval of WHO in May 2015 and the certification from Conformite Europeenne (CE) since 2010. Five of them were approved by Chinese Food and Drug
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Administration (CFDA), including Daan gene, ZJ BioTech, Puruikang, Tianlong and HuaDa BioTech, and put into use in West Africa. The kit of Puruikang, which was applied by Chinese medical team, took the principal responsibility for testing Ebola
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virus in epidemic area. However, no evaluation for these commercial kits had been
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performed yet. In this circumstance, the National Center for Clinical Laboratories (NCCL) conducted the assessment of seven Chinese commercial EBOV real time
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RT-PCR assays using virus-like particles, concerning the analytical sensitivity and
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precision for the qualitative diagnostic reagents.
MATERIALS AND METHODS Preparation of EBOV VLPs The full-length sequences of 3’ long terminal repeat (LTR) (1-55bp), NP (56-2340bp), and parts of GP (6291-6500bp and 7761-7850bp), L (13051-13710bp) and 5’ LTR (18271-18630bp) of recently epidemic Zaire strain (GenBank: KJ660346, KJ660347 and KJ660348) were gene synthesized by Thermo Fisher Scientific
ACCEPTED MANUSCRIPT Inc.(Beijing, China). The selected sequences were split into two parts and each part was individually subcloned into the vector of pACYC-MS2. The recombinant
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plasmids of pACYC-MS2-NP and pACYC-MS2-GP/L were transformed into the Escherichia coli strain BL21 (DE3). Two kinds of MS2 virus-like particles (VLPs)
sequences
along with
5’LTR
were
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respectively contained the RNA of NP fragment along with 3’LTR, and GP and L prepared
by inducible expressions
of
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isopropyl--D-thiogalactopyranoside (IPTG) and purification by means of exclusion chromatography according to our previous protocols (Sun Y et al., 2013; Song L et al., 2011).
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The prepared VLPs were digested with DNase and RNase in order to eliminate the
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influence of nucleic acid out of the capsid of VLPs. Subsequently, the contents were extracted from the VLPs using the kit for viral nucleic acid extraction (QIAGEN
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China Co., Ltd., Shanghai, China). Reverse transcription PCR (RT-PCR) and ordinary
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PCR were conducted simultaneously to verify the target RNA (not DNA) encapsulated in VLPs, which showed in S1 Fig. (Supplemental metarial). After extracted the RNA in successfully prepared VLPs, the concentration was tested and estimated following the formula: (6.02×1023 copies/mol) × armored RNA mass (g/ml)/molar mass (g/mol) = concentration (copies/ml). Two kinds of VLPs were diluted individually by normal human serum as substitutes of clinical samples. The characteristics of seven commercial kits In all of participated assays in this evaluation, the CFDA had approved the kits from Daan gene, ZJ BioTech, Puruikang in green channel for direct application in
ACCEPTED MANUSCRIPT West Africa. And the kit of Puruikang was appointed as the specialized detection assay for Chinese medical team. Subsequently, Huada and Tianlong were acquired the
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qualification from CFDA. Meanwhile, the manufacturers of Sansure and Kehua developed the EBOV detection kits for alternative productions. The characteristics of
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the seven commercial EBOV real-time RT-PCR detection kits are summarized in Table 1. Among all of the manufacturers for Zaire species detection, only Sansure
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preferred the magnetic-bead based method for RNA extraction and provided related reagents by themselves. Others took the conventional approach of spin column and QIAamp viral RNA mini kit (QIAGEN China Co., Ltd., Shanghai, China) was
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selected in the process for nucleic acid extraction. Meanwhile, Ebola virus types
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testing could merely been carried out by the kit of Sansure, while others focused on the specific detection of Zaire species. The manufacturer of Puruikang chose GP
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fragment as detection site while others mainly concentrated on NP site, which was
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reflected in figure 1.
TABLE 1 Characteristics of seven commercial EBOV real-time RT-PCR kits Commercial kits
Target region
Genotyping
Principle
Dye
IC a (dye)
Quantitative
RNA extracted method
Daan Gene ZJ BioTech
EBOV-NP EBOV- NP, 3’LTR
No No
TaqMan TaqMan
VIC FAM
Yes(CY5) Yes(HEX)
No No
spin column spin column
HuaDa BioTech KeHua
EBOV-NP EBOV-NP
No No
TaqMan TaqMan
FAM FAM
No No
spin column spin column
TianLong
EBOV-NP
No
TaqMan
FAM
No
spin column
Sansure
EBOV-NP
Yes
TaqMan
FAM
Yes(VIC) Yes(HEX or VIC) Yes(HEX or VIC) Yes(HEX)
No
magnetic-bead
ACCEPTED MANUSCRIPT SUDV-NP TAFV-GP BDBV-NP EBOV-GP
Puruikang
b
TaqMan
FAM
No
T
IC, Internal control.
Yes(HEX)
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a
No
Approval agency: CE, Conformite Europeenne; WHO, World Health Organization; CFDA,
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China’s Food and Drug Administration.
commercial assays. Ebola
virus
genome
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Fig.1 Organization of Ebola virus genome and detected target regions for
is
arranged
3’LTR-NP-VP35-VP40-GP-VP30-VP24-L-5’LTR.
in The
the
follow
detection
assays
order: were
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substituted by letters. A: ZJ BioTech; B: Daan Gene; C: HuaDa BioTech; D:
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Puruikang; E: TianLong; F: Sansure; G: Kehua. The detection regions for the seven
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commercial kits were marked in the sketch map.
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Limit of detection
Two kinds of EBOV VLPs were respectively diluted in a 10-fold series and tested preliminary from 107 copies/mL to 10 copies/mL. Applied biosystems 7500 fast real-time PCR system was used for evaluation of seven commercial kits. After the general scope of effective detection was determined, the endpoints for limitation were set. A series of diluted endpoints (500 copies/mL, 250 copies/mL, 100 copies/mL, 50 copies/mL, 25 copies/mL and 5 copies/mL) were used to compare the different concentrations for limit of detection (LOD) studies. Meanwhile, the logarithmic transformation for copies was conducted correspondingly. The experiments for LOD
spin column
ACCEPTED MANUSCRIPT were performed in five operating days, and every endpoint tested for three replicates in each time. Totally, 15 results for LOD were obtained and each data had only two
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outcomes: detected or undetected (Table 2). The data was processed and Probit
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analysis was used for regression analysis by SPSS version 19.0.
Aa
Parameter
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a
B
C
D
E
F
G
15/15 100 8/15 53.33 5/15 33.33 3/15 20 UN b UN 677 399~1961
15/15 100 15/15 100 14/15 93.33 12/15 80 10/15 66.67 1/15 6.67 99 61~246
15/15 100 15/15 100 12/15 80 9/15 60 1/15 6.67 UN 139 97~318
15/15 100 15/15 100 13/15 86.67 10/15 66.67 2/15 13.33 UN 119 86~255
15/15 100 15/15 100 15/15 100 13/15 86.67 8/15 53.33 1/15 6.67 77 50~176
15/15 100 15/15 100 14/15 93.33 10/15 66.67 3/15 20 UN 104 73~244
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15/15 100 15/15 100 15/15 100 15/15 100 13/15 86.67 3/15 20 33 21~85
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500 No. of positive/Total 2.70 % Positive 250 No. of positive/Total 2.40 % Positive 100 No. of positive/Total 2.00 % Positive 50 No. of positive/Total 1.70 % Positive 25 No. of positive/Total 1.40 % Positive 5 No. of positive/Total 0.70 % Positive Estimated value of 95% CI c Limits of 95% CI
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Copies/ml Log10
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TABLE 2 Limit of detection of seven commercial EBOV detection assays
The commercial assays participated in evaluation: A: Daan Gene; B: ZJ BioTech; C: HuaDa
BioTech; D: KeHua; E: TianLong; F: Sansure; G: Puruikang. b
UN, undetected.
c
CI, confidential interval.
Precision Combined with the results of LOD, the concentrations for precision detection were
ACCEPTED MANUSCRIPT confirmed accordingly. The VLP encapsulated GP fragment, with the concentrations of 105 copies/mL, 103 copies/mL and 110 copies/mL, was used for the assessment of
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the kit of Puruikang. Meanwhile, the serial concentration of 8×104 copies/mL, 8×102 copies/mL and 120 copies/mL for VLPs contained NP fragment were set to evaluate
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the remaining six kits. However, for the kit of ZJ Bio Tech, the lowest detection concentration was not included because of its high value of LOD. EBOV VLPs were
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diluted in advance and stored in 4℃ during the whole process for precision detection. The repeatability and reproducibility of the seven commercial assays were individually completed by detecting the intra-assay and inter-assay variations for the
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crossing threshold (Ct) values. The intra-assay precision was determined from 20
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replicates, while the inter-assay precision was calculated from six independent PCR runs with three replicates per run for each kit. Additionally, for the assay from Sansure
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Bio-Tech Co. Ltd., the precision was only detected with the Zaire strain of Ebola virus
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instead of other three types. The data was analyzed and the means, standard deviations (SD) and coefficient of variation (CV) were calculated by SPSS version 19.0. The accredited range of CV for intra-assay and inter-assay variations of all evaluated assays was below 5%.
RESULTS Evaluation of EBOV VLPs The MS2 VLPs encapsulated with target EBOV RNA sequences were prepared
ACCEPTED MANUSCRIPT successfully. Target fragments were correctly amplified by RT-PCR instead of ordinary PCR, which effectively proved the contents encapsulated in VLPs were RNA
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but not DNA after nucleotides extracted (S1 Fig.). The concentrations of two kinds of VLPs were both from 8×1010 copies/ml to 8×1011 copies/ml. The VLPs were stable in
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4℃ for two mouths which were consistent with the previous results (Song L et al., 2011). Seven commercial EBOV real-time RT-PCR kits were used and the scopes of
Limit of detection analysis
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dilution for VLPs were preliminarily determined.
Among the seven commercial EBOV real-time RT-PCR kits, the assay from
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Puruikang was evaluated by one of the VLPs encapsulated with GP fragment, while
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other kits by another VLP with the sequences of 3’LTR and NP. Generally, all of the seven reagents could detect EBOV under 1000 copies/mL and the details were list in
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Table 2. The Daan gene EBOV RT-PCR assay indicated the highest sensitivity, with a
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95% detection limit of 33 copies/mL (95% confidence interval (CI), 21 to 85 copies/mL), followed by the Sansure kit with 77 copies/mL (95% CI, 50 to 176 copies/mL) and the HuaDa BioTech kit with 99 copies/mL (95% CI, 61 to 246 copies/mL). The LOD of Kehua and Tianlong were 139 copies/mL (95% CI, 97 to 318 copies/mL) and 119 copies/mL (95% CI, 86 to 255 copies/mL) respectively, which were both more than 100 copies/mL. The ZJ BioTech kit possessed the maximum LOD in the seven different kits of 677 copies/mL (95% CI, 399 to 1961 copies/mL). However, the LOD of ZJ BioTech was also satisfied with the condition under 1000 copies/mL. The Puruikang kit could test the EBOV of 104 copies/mL
ACCEPTED MANUSCRIPT (95% CI, 73 to 244 copies/mL) for GP fragment. Precision
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In order to analyze the repeatability and reproducibility of the seven kits, intra-assay and inter-assay variations for the Ct values were demonstrated in Table 3.
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Referring the high concentration (NP: 8×104 copies/mL and GP: 105 copies/mL) of VLP for precision testing, the kit of Kehua demonstrated the maximum of CV
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(intra-assay: 2.01% and inter-assay: 3.84%) while the CV of the other kits were all below 2%. The relatively small variations were obtained by the reagent from ZJ Bio Tech (intra-assay CV: 0.30% and inter-assay CV: 0.98%) and Sansure (intra-assay CV:
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0.77% and inter-assay CV: 0.85%). The results in middle concentration of precision
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evaluation were similar to the high concentration group. In the group of low concentration, the relatively high values of CV were obtained by Kehua and Tianlong,
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while other kits were all lower than 3%. The maximum value of CV acquired by
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Kehua was 5.17%, which was the only one over 5% in the whole precision detection.
TABLE 3 Precision testing of different EBOV real-time RT-PCR kits
Concentration of VLPs a (Copies/mL)
Parameter b
Daan Gene
ZJ BioTech
HuaDa BioTech
KeHua
TianLong
Sansure
Puruikang
NP: 8×104 GP: 105
Intra-assay
28.350.19
29.950.09
27.470.32
21.420.43
22.130.40
27.200.21
24.010.19
(mean Ct S.D) CV (%)
0.67
0.30
1.16
2.01
1.81
0.77
0.79
Inter-assay
28.340.47
29.550.29
27.160.41
21.860.84
22.180.41
26.970.23
24.200.27
(mean Ct S.D) CV (%)
1.66
0.98
1.51
3.84
1.85
0.85
1.12
Intra-assay
35.170.59
34.280.62
33.540.40
28.830.98
28.880.74
34.050.46
30.960.39
NP: 800 GP: 103
(mean Ct S.D)
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3.40
2.56
1.35
1.26
Inter-assay (mean Ct S.D) CV (%)
34.950.48
34.320.72
33.080.68
28.491.41
28.780.86
33.840.57
31.300.45
1.37
2.10
2.06
4.95
2.99
1.68
1.44
Intra-assay
37.250.98
Undetected
35.450.46
31.561.01
36.550.40
34.580.80
(mean Ct S.D) CV (%)
2.63
-
1.30
Inter-assay
37.910.96
Undetected
35.390.79
(mean Ct S.D) CV (%)
2.53
-
2.23
T
1.81
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31.071.03
3.20
3.32
1.09
2.31
31.941.65
31.221.35
36.990.99
35.110.81
4.32
2.68
2.31
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a
1.68
5.17
VLPs with GP fragment was used for the assessment of the kit of Puruikang; VLPs with NP
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NP: 120 GP: 110
CV (%)
fragment was use for evaluating the other six assays.
Parameter, SD, standard deviations; CV, coefficient of variation.
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b
DISCUSSION
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In response to the outbreak of Ebolavirus (Zaire) in West Africa by 2014, a variety
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of diagnostic real time PCR reagents were manufactured subsequently in China. As the kits for qualitative detection, the parameters of precision, analytical sensitivity, accuracy and the detection ability for the serum samples panel were necessary for the performance evaluation. However, clinical samples had not been transported to China for strong contagiosity and high mortality of EBOV which was classified as the pathogen of BSL 4 agents. Without the positive samples, we could only test two of the most essential indicators, analytical sensitivity and precision, for qualitative kits assessment in this study. Meanwhile, in order to evaluate all of the kits in uniform standards, the same template for Zaire epidemic strain was selected and packaged in
ACCEPTED MANUSCRIPT VLPs as mimic. For analytical sensitivity, when the concentration was less than 50 copies/ml only
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the kit of Daan gene could ensure the effective detection, which indicated Daan gene was the most sensitive among all of the participated assays. The sensible capability of
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Sansure was followed closely with Daan gene. There was no significant difference of the LOD between Huada, Puruikang, Tianlong and Kehua. The maximum for
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analytical sensitivity was acquired by ZJ BioTech, which demonstrated the inferior detection capability in the seven different kits. Concerned about the precision, the maximum for SD (Ct value: 1.65) and CV (5.17%) were both acquired by Kehua,
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which was just over the requirement of 5% and indicated that there was still room to
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improve. In contrast, it was worth to praise for the good performance of ZJ BioTech and Sansure, especially in the evaluation with high concentration of VLPs. The
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remaining kits were similar in the precision evaluation of three different
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concentrations.
Overall, Daan gene had superior stability and sensitivity for EBOV detection in the seven kits. The performance of the kits from Puruikang, Huada and Sansure were creditable in all participated assays. For ZJ BioTech, there was notable reproducibility and tolerableness LOD. The repeatability and reproducibility for Kehua was improvable. Additionally, Tianlong kits achieved acceptable results in the evaluation of precision and analytical sensitivity. As the matter of fact, all of the seven commercial kits could meet the general requirement of LOD＜1000 copies/mL. In clinical work, the viral load in plasma of
ACCEPTED MANUSCRIPT EVD patients was often no less than 105 copis/mL (Schieffelin JS et al., 2014). It was usually seemed as negative results when the viral load of Ebola was less than 1000
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copies/mL (Kreuels B et al., 2014). Moreover, the outcome of real time PCR could only be one of valid evidences. The onset symptoms should be taken into
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consideration for the diagnosis of EVD (Saijo M et al., 2014). Therefore, all of the participated assays were eligible for Zaire species of Ebolavirus clinical detection. To
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reflect the repeatability and reproducibility, the intra-assay and inter-assay precision were tested. The requirement of CV＜5% was satisfied by six kits and Kehua in the concentration of more than 800 copies/mL, which supplied the trustworthy
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prerequisite for accuracy.
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In our study, VLPs took the place of positive samples and was adopted for kit evaluation. As an alternative, the selected fragments and target sites encapsulated in
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VLPs were the principal element for consideration. It was a guarantee for successful
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detection when the primers could be paired with the target sequences in VLPs. For EBOV detection, the sites of NP (Towner JS et al., 2004; Huang Y et al., 2012), GP (Trombley AR et al., 2010), L (Gibb TR et al., 2001) and 5’ LTR (Kurosaki Y et al., 2007) were the most conventional choices for primers design in previous research. In combination with the diagnostic condition in 2014 (Baize Sb et al., 2014), the whole length of 3’ LTR and NP, along with parts of GP, L and 5’ LTR of the epidemic strain were all designed to enfold in VLPs. This is the first time to conduct the reagent evaluation with VLPs encapsulated target RNA. Though VLPs as the substitution do have some disadvantages, such as
ACCEPTED MANUSCRIPT the high variability of RNA virus could not be reflected and the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for
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kits could not been evaluated. However, the advantages far outweigh its disadvantages. First, the structure of VLPs, with the capsid of MS2 bacteriophage and targeted RNA
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fragment, is similar to the real RNA virus, which could implement the complete simulation for viral detection. Second, it is flexible and optional for targeted RNA
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fragment encapsulated into VLPs. Third, VLPs is stable in room temperatures for a long time. Fourth, the biological hazard of the real virus could be neglected because there hardly any danger of MS2 VLP for human under normal condition. Fifth, the
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preparative technique of VLPs is mature and feasible. Additionally, the source of
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target fragment in VLPs is clearly which confirms that it could be used into the related evaluation work. Hence, VLPs could replace the clinical samples for kits evaluation
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and the relevant results are credible and guaranteed.
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS We thank Huiqing Li from Shandong Academy of Medical Sciences for instructing
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Probit Analysis with SPSS. We also thank all of the reagent manufacturers that provided the real-time RT-PCR kits for EBOV detection in this study.
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This work was supported by the National High Technology Research and Development Program of China 863 program (grant No. 2011AA02A116) and “AIDS
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China (No. 2013ZX10004805).
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and hepatitis, and other major infectious disease control and prevention” Program of
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ML, Branco LM, Vandi MA, Grant DS, Happi C, Gevao SM, Fletcher TE, Fowler RA,Bausch DG, Sabeti PC, Khan SH, Garry RF; KGH Lassa Fever Program; Viral Hemorrhagic Fever Consortium; WHO Clinical Response Team (2014) Clinical illness and outcomes in patients with Ebola in Sierra Leone. N Engl J Med
ACCEPTED MANUSCRIPT 371:2092-2100. Song L, Sun S, Li B, Pan Y, Li W, Zhang K, Li J (2011) External quality
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assessment for enterovirus 71 and coxsackievirus A16 detection by reverse transcription-PCR using armored RNA as a virus surrogate. J Clin Microbiol
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49:3591-3595.
Sun Y, Jia T, Sun Y, Han Y, Wang L, Zhang R, Zhang K, Lin G, Xie J, Li J. 2013. (H7N9) Virus
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External quality assessment for Avian Influenza A
detection
using
armored RNA. J Clin Microbiol 51:4055-4059.
Towner JS, Rollin PE, Bausch DG, Sanchez A, Crary SM, Vincent M, Lee
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WF, Spiropoulou CF, Ksiazek TG, Lukwiya M, Kaducu F, Downing R, Nichol ST
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(2004) Rapid diagnosis of Ebola hemorrhagic fever by reverse transcripition-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome. J
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Virol 78:4330-4341.
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Trombley AR, Wachter L, Garrison J, Buckley-Beason VA, Jahrling J, Hensley LE, Schoepp RJ, Norwood DA, Goba A, Fair JN, Kulesh DA (2010) Compregensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses and New World hantavirused. Am J Trop Med Hyg 82:954-960. World Health Organization (2014) Ebola virus disease: fact sheet no. 103. Available at http://www.who.int/mediacentre/factsheets/fs103/en.
ACCEPTED MANUSCRIPT Fig.1 Organization of Ebola virus genome and detected target regions for
Ebola
virus
genome
is
arranged
in
The
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3’LTR-NP-VP35-VP40-GP-VP30-VP24-L-5’LTR.
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commercial assays.
the
follow
detection
assays
order: were
substituted by letters. A: ZJ BioTech; B: Daan Gene; C: HuaDa BioTech; D:
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Puruikang; E: TianLong; F: Sansure; G: Kehua. The detection regions for the seven
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CE
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commercial kits were marked in the sketch map.
ACCEPTED MANUSCRIPT Highlights Several real-time PCR kits for Zaire Ebolavirus have been evaluated in China.
Viruses like particles encapsulated EBOV RNA have been prepared as positive
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substitutes.
The limit of detection and the precision were completed for the assays.
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Requirements for clinical EBOV detection are basically satisfied except
It is feasible for viruses like particles as a mimic of positive samples in
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assays evaluation.
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one reagent.
S0732-8893(15)00289-8 doi: 10.1016/j.diagmicrobio.2015.07.025 DMB 13885
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
14 May 2015 8 July 2015 30 July 2015
Please cite this article as: Wang Guojing, Zhang Rui, Chang Le, Zhang Dong, Zhang Lei, Yang Xin, Liu Chao, Ding Jiansheng, Zhang Kuo, Lin Guigao, Jiehong Xie, Li Jinming, The evaluation of seven commercial real-time PCR kits for Zaire Ebolavirus using viruslike particles encapsulated EBOV RNA, Diagnostic Microbiology and Infectious Disease (2015), doi: 10.1016/j.diagmicrobio.2015.07.025
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ACCEPTED MANUSCRIPT Title page The evaluation of seven commercial real-time PCR kits for Zaire
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Ebolavirus using virus-like particles encapsulated EBOV RNA Guojing Wang1, 2, Rui Zhang1, Le Chang1, 2, Dong Zhang1, 2, Lei Zhang1, 3, Xin
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Yang1, 2, Chao Liu1, 2, Jiansheng Ding1, 2, Kuo Zhang1, Guigao Lin1, Xie Jiehong1,
1
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Jinming Li1*
National Center for Clinical Laboratories, Beijing Hospital of National Health and
Family Planning Commission, Beijing, People’s Republic of China Graduate School, Peking Union Medical College, Chinese Academy of Medical
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2
Fifth School of Clinical Medicine, Peking University, Beijing, People’s Republic of
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China
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3
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Sciences, Beijing, People’s Republic of China
*Corresponding author Mailing address: National Center for Clinical Laboratories Beijing Hospital, No.1 Dahua Road, Dongdan, Beijing 100730, P R China. Tel: 86-10-58115053; Fax: 86-10-65212064 E-mail: [email protected] Running title: Evaluation of EBOV real-time RT-PCR kits Word counts of the abstract: 143 words Word counts of the body of the text: 2539 words
ACCEPTED MANUSCRIPT
ABSTRACT
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Several Chinese commercial real-time PCR kits for Zaire Ebolavirus have been developed after the Ebola outbreak and used by Chinese medical teams in West
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Africa. In order to know the essential performance indicators of these kits, analytical sensitivity and precision were evaluated with virus-like particles (VLPs)
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encapsulated EBOV RNA. The limit of detection and the precision were completed with a series of VLPs. The maximum and minimum of limit of detection was acquired by ZJ BioTech and Daan gene respectively. For precision, all of the
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detection results were less than 5% except the maximum 5.17%. Among them
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Puruikang, Daan gene, Sansure, ZJ BioTech and Huada demonstrated superior reproducibility. Overall, the requirements of LOD＜1000 copies/mL and CV＜5%
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could be satisfied by all kits except Kehua. Meanwhile, it is feasible for VLPs as a
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substitute of positive samples in assays evaluation.
Keywords: Ebola virus, kits evaluation, real time RT-PCR, virus-like particles.
ACCEPTED MANUSCRIPT INTRODUCTION Ebola virus belongs to the Filoviridae family and contains a single negative-sense,
phosphoprotein
(VP35),
matrix
protein
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nearly 19kb RNA strand which encodes seven proteins: nucleoprotein (NP), (VP40),
glycoprotein
(GP),
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replication-transcription protein (VP30), matrix protein (VP24) and RNA dependent RNA polymerase (L) (Fields BN et al., 2007). Among the five species (Bundibugyo,
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Sudan, Zaire, Tai Forest and Reston), the Zaire species has the most frequent outbreaks and the highest mortality rate from 57% to 90% (McElroy AK et al., 2014) to human being. In 2014, viral isolation and sequence alignment were conducted and
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(Baize S et al., 2014).
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identified that the epidemic strain in West Africa was Zaire Ebolavirus (EBOV)
In early August, 2014, the World Health Organization (WHO) had announced the
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outbreak of Ebola virus disease (EVD) in West Africa as “public healthy emergency
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of international concern”. The number of both infectors and deaths of EVD in recent epidemic was the highest and more than 22 prior outbreaks in total since the firstly identified in 1976 (WHO, 2014). The center for EBOV plague was Guinea, Liberia, and Sierra Leone, along with other countries in West Africa, such as Nigeria and Senegal. Europe and the United States had also been reported as sporadic affected regions (Ledgerwood JE et al., 2014). Confronting with the situation above, China authority had provided supports for Africa since September 2014. Sierra Leone - China friendship hospital was built and a mobile biosafety level-3 laboratory (BSL-3), diagnostic reagents for EBOV, public
ACCEPTED MANUSCRIPT health experts and medical staffers were all transported and dispatched for four times to EBOV-affected areas (Gao GF et al., 2014). In response to the aid of West Africa,
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plenty of commercial kits for qualitative detecting Zaire Ebola virus had been independently developed in China.. The ZJ BioTech kit had obtained the fully
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approval of WHO in May 2015 and the certification from Conformite Europeenne (CE) since 2010. Five of them were approved by Chinese Food and Drug
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Administration (CFDA), including Daan gene, ZJ BioTech, Puruikang, Tianlong and HuaDa BioTech, and put into use in West Africa. The kit of Puruikang, which was applied by Chinese medical team, took the principal responsibility for testing Ebola
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virus in epidemic area. However, no evaluation for these commercial kits had been
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performed yet. In this circumstance, the National Center for Clinical Laboratories (NCCL) conducted the assessment of seven Chinese commercial EBOV real time
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RT-PCR assays using virus-like particles, concerning the analytical sensitivity and
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precision for the qualitative diagnostic reagents.
MATERIALS AND METHODS Preparation of EBOV VLPs The full-length sequences of 3’ long terminal repeat (LTR) (1-55bp), NP (56-2340bp), and parts of GP (6291-6500bp and 7761-7850bp), L (13051-13710bp) and 5’ LTR (18271-18630bp) of recently epidemic Zaire strain (GenBank: KJ660346, KJ660347 and KJ660348) were gene synthesized by Thermo Fisher Scientific
ACCEPTED MANUSCRIPT Inc.(Beijing, China). The selected sequences were split into two parts and each part was individually subcloned into the vector of pACYC-MS2. The recombinant
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plasmids of pACYC-MS2-NP and pACYC-MS2-GP/L were transformed into the Escherichia coli strain BL21 (DE3). Two kinds of MS2 virus-like particles (VLPs)
sequences
along with
5’LTR
were
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respectively contained the RNA of NP fragment along with 3’LTR, and GP and L prepared
by inducible expressions
of
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isopropyl--D-thiogalactopyranoside (IPTG) and purification by means of exclusion chromatography according to our previous protocols (Sun Y et al., 2013; Song L et al., 2011).
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The prepared VLPs were digested with DNase and RNase in order to eliminate the
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influence of nucleic acid out of the capsid of VLPs. Subsequently, the contents were extracted from the VLPs using the kit for viral nucleic acid extraction (QIAGEN
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China Co., Ltd., Shanghai, China). Reverse transcription PCR (RT-PCR) and ordinary
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PCR were conducted simultaneously to verify the target RNA (not DNA) encapsulated in VLPs, which showed in S1 Fig. (Supplemental metarial). After extracted the RNA in successfully prepared VLPs, the concentration was tested and estimated following the formula: (6.02×1023 copies/mol) × armored RNA mass (g/ml)/molar mass (g/mol) = concentration (copies/ml). Two kinds of VLPs were diluted individually by normal human serum as substitutes of clinical samples. The characteristics of seven commercial kits In all of participated assays in this evaluation, the CFDA had approved the kits from Daan gene, ZJ BioTech, Puruikang in green channel for direct application in
ACCEPTED MANUSCRIPT West Africa. And the kit of Puruikang was appointed as the specialized detection assay for Chinese medical team. Subsequently, Huada and Tianlong were acquired the
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qualification from CFDA. Meanwhile, the manufacturers of Sansure and Kehua developed the EBOV detection kits for alternative productions. The characteristics of
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the seven commercial EBOV real-time RT-PCR detection kits are summarized in Table 1. Among all of the manufacturers for Zaire species detection, only Sansure
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preferred the magnetic-bead based method for RNA extraction and provided related reagents by themselves. Others took the conventional approach of spin column and QIAamp viral RNA mini kit (QIAGEN China Co., Ltd., Shanghai, China) was
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selected in the process for nucleic acid extraction. Meanwhile, Ebola virus types
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testing could merely been carried out by the kit of Sansure, while others focused on the specific detection of Zaire species. The manufacturer of Puruikang chose GP
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fragment as detection site while others mainly concentrated on NP site, which was
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reflected in figure 1.
TABLE 1 Characteristics of seven commercial EBOV real-time RT-PCR kits Commercial kits
Target region
Genotyping
Principle
Dye
IC a (dye)
Quantitative
RNA extracted method
Daan Gene ZJ BioTech
EBOV-NP EBOV- NP, 3’LTR
No No
TaqMan TaqMan
VIC FAM
Yes(CY5) Yes(HEX)
No No
spin column spin column
HuaDa BioTech KeHua
EBOV-NP EBOV-NP
No No
TaqMan TaqMan
FAM FAM
No No
spin column spin column
TianLong
EBOV-NP
No
TaqMan
FAM
No
spin column
Sansure
EBOV-NP
Yes
TaqMan
FAM
Yes(VIC) Yes(HEX or VIC) Yes(HEX or VIC) Yes(HEX)
No
magnetic-bead
ACCEPTED MANUSCRIPT SUDV-NP TAFV-GP BDBV-NP EBOV-GP
Puruikang
b
TaqMan
FAM
No
T
IC, Internal control.
Yes(HEX)
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a
No
Approval agency: CE, Conformite Europeenne; WHO, World Health Organization; CFDA,
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China’s Food and Drug Administration.
commercial assays. Ebola
virus
genome
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Fig.1 Organization of Ebola virus genome and detected target regions for
is
arranged
3’LTR-NP-VP35-VP40-GP-VP30-VP24-L-5’LTR.
in The
the
follow
detection
assays
order: were
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substituted by letters. A: ZJ BioTech; B: Daan Gene; C: HuaDa BioTech; D:
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Puruikang; E: TianLong; F: Sansure; G: Kehua. The detection regions for the seven
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commercial kits were marked in the sketch map.
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Limit of detection
Two kinds of EBOV VLPs were respectively diluted in a 10-fold series and tested preliminary from 107 copies/mL to 10 copies/mL. Applied biosystems 7500 fast real-time PCR system was used for evaluation of seven commercial kits. After the general scope of effective detection was determined, the endpoints for limitation were set. A series of diluted endpoints (500 copies/mL, 250 copies/mL, 100 copies/mL, 50 copies/mL, 25 copies/mL and 5 copies/mL) were used to compare the different concentrations for limit of detection (LOD) studies. Meanwhile, the logarithmic transformation for copies was conducted correspondingly. The experiments for LOD
spin column
ACCEPTED MANUSCRIPT were performed in five operating days, and every endpoint tested for three replicates in each time. Totally, 15 results for LOD were obtained and each data had only two
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outcomes: detected or undetected (Table 2). The data was processed and Probit
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analysis was used for regression analysis by SPSS version 19.0.
Aa
Parameter
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a
B
C
D
E
F
G
15/15 100 8/15 53.33 5/15 33.33 3/15 20 UN b UN 677 399~1961
15/15 100 15/15 100 14/15 93.33 12/15 80 10/15 66.67 1/15 6.67 99 61~246
15/15 100 15/15 100 12/15 80 9/15 60 1/15 6.67 UN 139 97~318
15/15 100 15/15 100 13/15 86.67 10/15 66.67 2/15 13.33 UN 119 86~255
15/15 100 15/15 100 15/15 100 13/15 86.67 8/15 53.33 1/15 6.67 77 50~176
15/15 100 15/15 100 14/15 93.33 10/15 66.67 3/15 20 UN 104 73~244
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15/15 100 15/15 100 15/15 100 15/15 100 13/15 86.67 3/15 20 33 21~85
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500 No. of positive/Total 2.70 % Positive 250 No. of positive/Total 2.40 % Positive 100 No. of positive/Total 2.00 % Positive 50 No. of positive/Total 1.70 % Positive 25 No. of positive/Total 1.40 % Positive 5 No. of positive/Total 0.70 % Positive Estimated value of 95% CI c Limits of 95% CI
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Copies/ml Log10
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TABLE 2 Limit of detection of seven commercial EBOV detection assays
The commercial assays participated in evaluation: A: Daan Gene; B: ZJ BioTech; C: HuaDa
BioTech; D: KeHua; E: TianLong; F: Sansure; G: Puruikang. b
UN, undetected.
c
CI, confidential interval.
Precision Combined with the results of LOD, the concentrations for precision detection were
ACCEPTED MANUSCRIPT confirmed accordingly. The VLP encapsulated GP fragment, with the concentrations of 105 copies/mL, 103 copies/mL and 110 copies/mL, was used for the assessment of
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the kit of Puruikang. Meanwhile, the serial concentration of 8×104 copies/mL, 8×102 copies/mL and 120 copies/mL for VLPs contained NP fragment were set to evaluate
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the remaining six kits. However, for the kit of ZJ Bio Tech, the lowest detection concentration was not included because of its high value of LOD. EBOV VLPs were
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diluted in advance and stored in 4℃ during the whole process for precision detection. The repeatability and reproducibility of the seven commercial assays were individually completed by detecting the intra-assay and inter-assay variations for the
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crossing threshold (Ct) values. The intra-assay precision was determined from 20
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replicates, while the inter-assay precision was calculated from six independent PCR runs with three replicates per run for each kit. Additionally, for the assay from Sansure
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Bio-Tech Co. Ltd., the precision was only detected with the Zaire strain of Ebola virus
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instead of other three types. The data was analyzed and the means, standard deviations (SD) and coefficient of variation (CV) were calculated by SPSS version 19.0. The accredited range of CV for intra-assay and inter-assay variations of all evaluated assays was below 5%.
RESULTS Evaluation of EBOV VLPs The MS2 VLPs encapsulated with target EBOV RNA sequences were prepared
ACCEPTED MANUSCRIPT successfully. Target fragments were correctly amplified by RT-PCR instead of ordinary PCR, which effectively proved the contents encapsulated in VLPs were RNA
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but not DNA after nucleotides extracted (S1 Fig.). The concentrations of two kinds of VLPs were both from 8×1010 copies/ml to 8×1011 copies/ml. The VLPs were stable in
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4℃ for two mouths which were consistent with the previous results (Song L et al., 2011). Seven commercial EBOV real-time RT-PCR kits were used and the scopes of
Limit of detection analysis
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dilution for VLPs were preliminarily determined.
Among the seven commercial EBOV real-time RT-PCR kits, the assay from
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Puruikang was evaluated by one of the VLPs encapsulated with GP fragment, while
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other kits by another VLP with the sequences of 3’LTR and NP. Generally, all of the seven reagents could detect EBOV under 1000 copies/mL and the details were list in
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Table 2. The Daan gene EBOV RT-PCR assay indicated the highest sensitivity, with a
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95% detection limit of 33 copies/mL (95% confidence interval (CI), 21 to 85 copies/mL), followed by the Sansure kit with 77 copies/mL (95% CI, 50 to 176 copies/mL) and the HuaDa BioTech kit with 99 copies/mL (95% CI, 61 to 246 copies/mL). The LOD of Kehua and Tianlong were 139 copies/mL (95% CI, 97 to 318 copies/mL) and 119 copies/mL (95% CI, 86 to 255 copies/mL) respectively, which were both more than 100 copies/mL. The ZJ BioTech kit possessed the maximum LOD in the seven different kits of 677 copies/mL (95% CI, 399 to 1961 copies/mL). However, the LOD of ZJ BioTech was also satisfied with the condition under 1000 copies/mL. The Puruikang kit could test the EBOV of 104 copies/mL
ACCEPTED MANUSCRIPT (95% CI, 73 to 244 copies/mL) for GP fragment. Precision
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In order to analyze the repeatability and reproducibility of the seven kits, intra-assay and inter-assay variations for the Ct values were demonstrated in Table 3.
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Referring the high concentration (NP: 8×104 copies/mL and GP: 105 copies/mL) of VLP for precision testing, the kit of Kehua demonstrated the maximum of CV
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(intra-assay: 2.01% and inter-assay: 3.84%) while the CV of the other kits were all below 2%. The relatively small variations were obtained by the reagent from ZJ Bio Tech (intra-assay CV: 0.30% and inter-assay CV: 0.98%) and Sansure (intra-assay CV:
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0.77% and inter-assay CV: 0.85%). The results in middle concentration of precision
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evaluation were similar to the high concentration group. In the group of low concentration, the relatively high values of CV were obtained by Kehua and Tianlong,
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while other kits were all lower than 3%. The maximum value of CV acquired by
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Kehua was 5.17%, which was the only one over 5% in the whole precision detection.
TABLE 3 Precision testing of different EBOV real-time RT-PCR kits
Concentration of VLPs a (Copies/mL)
Parameter b
Daan Gene
ZJ BioTech
HuaDa BioTech
KeHua
TianLong
Sansure
Puruikang
NP: 8×104 GP: 105
Intra-assay
28.350.19
29.950.09
27.470.32
21.420.43
22.130.40
27.200.21
24.010.19
(mean Ct S.D) CV (%)
0.67
0.30
1.16
2.01
1.81
0.77
0.79
Inter-assay
28.340.47
29.550.29
27.160.41
21.860.84
22.180.41
26.970.23
24.200.27
(mean Ct S.D) CV (%)
1.66
0.98
1.51
3.84
1.85
0.85
1.12
Intra-assay
35.170.59
34.280.62
33.540.40
28.830.98
28.880.74
34.050.46
30.960.39
NP: 800 GP: 103
(mean Ct S.D)
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3.40
2.56
1.35
1.26
Inter-assay (mean Ct S.D) CV (%)
34.950.48
34.320.72
33.080.68
28.491.41
28.780.86
33.840.57
31.300.45
1.37
2.10
2.06
4.95
2.99
1.68
1.44
Intra-assay
37.250.98
Undetected
35.450.46
31.561.01
36.550.40
34.580.80
(mean Ct S.D) CV (%)
2.63
-
1.30
Inter-assay
37.910.96
Undetected
35.390.79
(mean Ct S.D) CV (%)
2.53
-
2.23
T
1.81
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31.071.03
3.20
3.32
1.09
2.31
31.941.65
31.221.35
36.990.99
35.110.81
4.32
2.68
2.31
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a
1.68
5.17
VLPs with GP fragment was used for the assessment of the kit of Puruikang; VLPs with NP
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NP: 120 GP: 110
CV (%)
fragment was use for evaluating the other six assays.
Parameter, SD, standard deviations; CV, coefficient of variation.
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b
DISCUSSION
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In response to the outbreak of Ebolavirus (Zaire) in West Africa by 2014, a variety
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of diagnostic real time PCR reagents were manufactured subsequently in China. As the kits for qualitative detection, the parameters of precision, analytical sensitivity, accuracy and the detection ability for the serum samples panel were necessary for the performance evaluation. However, clinical samples had not been transported to China for strong contagiosity and high mortality of EBOV which was classified as the pathogen of BSL 4 agents. Without the positive samples, we could only test two of the most essential indicators, analytical sensitivity and precision, for qualitative kits assessment in this study. Meanwhile, in order to evaluate all of the kits in uniform standards, the same template for Zaire epidemic strain was selected and packaged in
ACCEPTED MANUSCRIPT VLPs as mimic. For analytical sensitivity, when the concentration was less than 50 copies/ml only
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the kit of Daan gene could ensure the effective detection, which indicated Daan gene was the most sensitive among all of the participated assays. The sensible capability of
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Sansure was followed closely with Daan gene. There was no significant difference of the LOD between Huada, Puruikang, Tianlong and Kehua. The maximum for
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analytical sensitivity was acquired by ZJ BioTech, which demonstrated the inferior detection capability in the seven different kits. Concerned about the precision, the maximum for SD (Ct value: 1.65) and CV (5.17%) were both acquired by Kehua,
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which was just over the requirement of 5% and indicated that there was still room to
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improve. In contrast, it was worth to praise for the good performance of ZJ BioTech and Sansure, especially in the evaluation with high concentration of VLPs. The
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remaining kits were similar in the precision evaluation of three different
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concentrations.
Overall, Daan gene had superior stability and sensitivity for EBOV detection in the seven kits. The performance of the kits from Puruikang, Huada and Sansure were creditable in all participated assays. For ZJ BioTech, there was notable reproducibility and tolerableness LOD. The repeatability and reproducibility for Kehua was improvable. Additionally, Tianlong kits achieved acceptable results in the evaluation of precision and analytical sensitivity. As the matter of fact, all of the seven commercial kits could meet the general requirement of LOD＜1000 copies/mL. In clinical work, the viral load in plasma of
ACCEPTED MANUSCRIPT EVD patients was often no less than 105 copis/mL (Schieffelin JS et al., 2014). It was usually seemed as negative results when the viral load of Ebola was less than 1000
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copies/mL (Kreuels B et al., 2014). Moreover, the outcome of real time PCR could only be one of valid evidences. The onset symptoms should be taken into
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consideration for the diagnosis of EVD (Saijo M et al., 2014). Therefore, all of the participated assays were eligible for Zaire species of Ebolavirus clinical detection. To
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reflect the repeatability and reproducibility, the intra-assay and inter-assay precision were tested. The requirement of CV＜5% was satisfied by six kits and Kehua in the concentration of more than 800 copies/mL, which supplied the trustworthy
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prerequisite for accuracy.
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In our study, VLPs took the place of positive samples and was adopted for kit evaluation. As an alternative, the selected fragments and target sites encapsulated in
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VLPs were the principal element for consideration. It was a guarantee for successful
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detection when the primers could be paired with the target sequences in VLPs. For EBOV detection, the sites of NP (Towner JS et al., 2004; Huang Y et al., 2012), GP (Trombley AR et al., 2010), L (Gibb TR et al., 2001) and 5’ LTR (Kurosaki Y et al., 2007) were the most conventional choices for primers design in previous research. In combination with the diagnostic condition in 2014 (Baize Sb et al., 2014), the whole length of 3’ LTR and NP, along with parts of GP, L and 5’ LTR of the epidemic strain were all designed to enfold in VLPs. This is the first time to conduct the reagent evaluation with VLPs encapsulated target RNA. Though VLPs as the substitution do have some disadvantages, such as
ACCEPTED MANUSCRIPT the high variability of RNA virus could not be reflected and the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for
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kits could not been evaluated. However, the advantages far outweigh its disadvantages. First, the structure of VLPs, with the capsid of MS2 bacteriophage and targeted RNA
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fragment, is similar to the real RNA virus, which could implement the complete simulation for viral detection. Second, it is flexible and optional for targeted RNA
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fragment encapsulated into VLPs. Third, VLPs is stable in room temperatures for a long time. Fourth, the biological hazard of the real virus could be neglected because there hardly any danger of MS2 VLP for human under normal condition. Fifth, the
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preparative technique of VLPs is mature and feasible. Additionally, the source of
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target fragment in VLPs is clearly which confirms that it could be used into the related evaluation work. Hence, VLPs could replace the clinical samples for kits evaluation
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and the relevant results are credible and guaranteed.
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS We thank Huiqing Li from Shandong Academy of Medical Sciences for instructing
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Probit Analysis with SPSS. We also thank all of the reagent manufacturers that provided the real-time RT-PCR kits for EBOV detection in this study.
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This work was supported by the National High Technology Research and Development Program of China 863 program (grant No. 2011AA02A116) and “AIDS
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China (No. 2013ZX10004805).
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and hepatitis, and other major infectious disease control and prevention” Program of
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ACCEPTED MANUSCRIPT Fig.1 Organization of Ebola virus genome and detected target regions for
Ebola
virus
genome
is
arranged
in
The
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3’LTR-NP-VP35-VP40-GP-VP30-VP24-L-5’LTR.
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commercial assays.
the
follow
detection
assays
order: were
substituted by letters. A: ZJ BioTech; B: Daan Gene; C: HuaDa BioTech; D:
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Puruikang; E: TianLong; F: Sansure; G: Kehua. The detection regions for the seven
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commercial kits were marked in the sketch map.
ACCEPTED MANUSCRIPT Highlights Several real-time PCR kits for Zaire Ebolavirus have been evaluated in China.
Viruses like particles encapsulated EBOV RNA have been prepared as positive
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substitutes.
The limit of detection and the precision were completed for the assays.
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Requirements for clinical EBOV detection are basically satisfied except
It is feasible for viruses like particles as a mimic of positive samples in
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assays evaluation.
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one reagent.