JCM Accepts, published online ahead of print on 29 January 2014 J. Clin. Microbiol. doi:10.1128/JCM.00245-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Simultaneous Detection of Five Enteric Viruses Associated with Gastroenteritis
2
Using a PCR Assay: a Single Real-time Multiplex Reaction and its Clinical Application
3 4
Yixiang Jiang1*, Lin Fang3*, Xiaolu Shi2*, Hailong Zhang2, Yinghui Li2, Yinman Lin2, Yaqun
5
Qiu2, Qingliang Chen4, Hui Li3, Li Zhou2†, Qinghua Hu1,2†
6 7
Affiliation:
8
1
9
2
School of Life Sciences, Shenzhen University, Shenzhen 518000, Guangdong, China Shenzhen Major Infectious Disease Control Key Laboratory, Shenzhen Center for Disease
10
Control and Prevention, Shenzhen 518055, Guangdong Province, China
11
3
12
Guangdong Province, China
13
4
14
*These authors contributed equally to this work.
15
† Correspondence authors:
16
L. Zhou, Shenzhen Centre for Disease Control and Prevention, Shenzhen 518055, Guangdong
17
Province, China. Phone: +86-755-25623237, Fax: +86-755-25623237.
18
E-mail: [email protected]
19
Q. Hu, Shenzhen Centre for Disease Control and Prevention, Shenzhen 518055, Guangdong
20
Province, China. Phone: +86-755-86580705, Fax: +86-755-25532595.
21
E-mail: [email protected]
Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 518000,
School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China
1
22
Running Title: Single reaction of five enteric viruses by multiplex real-time PCR
23
Key words: viral gastroenteritis, multiplex real-time PCR, the reverse transcription, single
24
Word count: Abstract 47, Text 1141
2
25
Abstract
26
We developed a highly sensitive, reverse transcription and multiplex real-time PCR (rtPCR)
27
assay that can identify five viruses; including six genogroups, in a single reaction: Norovirus
28
genogroups I, II;, Sapovirus genogroups I, II, IV and V, human Rotaviruses A, Adenovirus
29
serotypes 40 and 41, and human Astrovirus. In comparison to monoplex rtPCR assays,
30
sensitivities and specificities of the multiplex rtPCR ranged 75-100% and 99-100%, respectively,
31
when evaluated on 812 clinical stool specimens.
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 3
47
Norovirus genogroups I, II (NV) (1, 2), Sapovirus genogroups I, II, IV and V (SaV) (3),
48
human Rotaviruses A (HRV) (4), Adenovirus serotypes 40 and 41 (AdV) (5), and human
49
Astrovirus (AstV) (6) are the most common cause of viral gastroenteritis and result in large
50
outbreak of viral diarrhea (7-11). Rapid and accurate detection methods are essential for response
51
to viral diarrhea outbreaks and management of the patients (12, 13). Current detection methods
52
include electron microscopy (14), enzyme immunoassay (EIA) (15)，conventional or real-time
53
PCR (16-19), or the Luminex xMAP technology (12-13). These methods are laborious (14) or
54
need post gel electrophoresis (18) or allow for simultaneous detection of three or four viral
55
pathogens in one reaction (16,17,19), or need opening of reaction tubes for hybridization(12-13).
56
Here, based on the principle of multicolor combinational probe coding (MCPC) (20, 21), we
57
developed a more effective multiplex real-time PCR (rtPCR) assay for the simultaneous detection
58
of five major enteric viruses in a single reaction.
59
All gene sequences retrieved from GenBank were aligned using Clustal W software. The
60
primers and modified molecular beacon probes based on the conserved regions were designed
61
using Primer Premier (version 5.0), Oligo (version 6.31) and DNA folding software
62
(http://mfold.ma.albany.edu/?q=mfold/dna-folding-form). The sequences of the primers for
63
NV and HRV were adapted from the literature (19, 22). The primers had a common tag sequence
64
at the 5’ ends to keep the formation of primer dimmers to a minimum. Equine Arteritis virus
65
(EAV) was employed as an internal control to monitor the inhibition of amplification (23).
66 67 68
All primers and probes were commercially synthesized, labeled and purified by the company Sangon Biotech (Shanghai, China) (Supplementary Table S1). Ten positive samples in which SaV, NV GI, NV GII, HRV, AdV and AstV were identified by 4
69
PCR with sequencing were used for the plasmid DNA standards preparation and initial multiplex
70
rtPCR assay development. Viral DNA/RNA of clinical stool samples was extracted using the
71
Roche High Pure Vial Nucleic Acid kit (Roche Diagnostics, Penzberg, Germany). For the reverse
72
transcription, 5μl of extracted RNA was transcribed into cDNA using Primer Script 1st strand
73
cDNA Synthesis Kit (Takara Biotechnology Company, Dalian, China).
74
Multiplex rtPCR were conducted using a BIO-RAD CFX96 real-time PCR system. The final
75
volume was 25μl, which included 1×PCR buffer, 3mM MgCl2, 2μl of deoxynucleoside
76
triphosphate (2.5 mM), and 1 unit Taq polymerase, 0.1 μM to 0.9 μM specific primers and probes
77
(Supplementary Table S1) and 5μl of cDNA or DNA template. Optimal cycling conditions
78
included a first stage initiated by heating at 95°C for 3 min, followed by a second stage of five
79
cycles of 95 °C for 10 s, 58 °C for 20 s, 72 °C for 15s, and finally by a third stage of 40 cycles of
80
95 °C for 10s, 55 °C for 32s, 72 °C for 15s. FAM, HEX, ROX, Cy5 and qursar705 fluorescence was
81
detected and recorded at each step of primer annealing during the third stage.
82 83 84 85 86 87 88 89 90
To confirm whether the target genes selected were specific for each virus, nucleic acids of Giardia lamblia, Entamoeba histolytica, Cryptosporidium paHRVum, Staphylococcus aureus, Shigella sp., Yersinia enterocolitica, Campylobacter jejuni and Human enterovirus 71 and ten enteric viruses positive samples were tested by multiplex rtPCR. No cross-reaction was observed and all five of the targeted genes were identified correctly by the multiplex rtPCR (Supplementary Figure S1), indicating that the developed assay had good specificity. The detection limits of the multiplex rtPCR assays were assessed twice using seven serial ten-fold dilutions (107–101 copies/μl) of the plasmid DNA standards and four serial ten-fold dilutions (106-103 copies/μl) of the plasmid DNA standards were prepared for the analysis of the intro-reproducibility in triplicate 5
91 92 93 94 95
experiments. The detection limits of the five viruses were 10 copies / μl. Amplification efficiency ranged from 87.6% to 99.2% with strong correlation coefficients (R2≥0.99) (Supplementary Table S2 and Figure S2). The CT values from the triplicate experiments differ within one and the coefficient of variation (CV) for each target virus ranged from 0.06% to 2.3%, which indicated that the assay was reproducible (Supplementary Table S3).
96
Due to the clinical samples often containing mixed infections (24-27), HRV and AdV were
97
used as the representative in conjunction to simulate co-infection. Each of the 2.5μl plasmid DNA
98
standards were combined in equal amounts ranging from 102 to 106 copies / μl for multiplex rtPCR.
99
HRV and AdV could be simultaneously detected apart from when the number of HRV copies was
100
10,000 times higher than the number of AdV copies, and this prevented the AdV from being
101
detected (Figure 1).
102
To further validate the multiplex rtPCR assay’s specificity and sensitivity, 812 clinical stool
103
samples were collected from ten hospitals in four cities in Guangdong province and were detected
104
using the newly developed assay with direct comparison with commercial monoplex rtPCR kits
105
(ZJ Bio-Tech, Shanghai, China ). All monoplex rtPCR positive products of clinical samples were
106
sequenced for the further confirmation. Among the 812 stool samples, 372 were virus positive.
107
From which 29 stool specimens were found to be co-infected with two viruses, which included
108
11of NV GII and AdV, 10 of HRV and AdV, five of NV GII and HRV, as well as three of NV GI
109
and NV GII (Table 1).
110
When compared with monoplex rtPCR, the sensitivity of the multiplex rtPCR assay ranged
111
from 75% to100% and the specificity ranged from 99% to100%. The agreement ranged from
112
97.8%-100% and the kappa correlation ranged from 0.85 to 1.0 (Table 1). 6
113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129
130 131 132 133 134 135
This study has the limitation that requires further improvement. Two NV GI samples, 10 HRV samples, One AdV sample and 12 NV GII samples were positive by monoplex rtPCR but negative by multiplex rtPCR, which was due to nucleic acid degradation. More clinical samples will be collected for the further validation. Among 812 stool samples collected between January 2012 and July 2013, 422 stool samples were collected from Shenzhen, of which 216 were positive (including 116 NV II, 65 HRV, 17 AdV, eight NV I, eight AstV and two SaV); 222 from Guangzhou including 39 NV II, 55 HRV, four AdV, one AstV and two SaV positive; 108 from Dongguan with 40 NV II, three HRV and two AdV positive; and 60 from Zhongshan having seven NV II and three HRV positive (Supplementary Table S4). The data showed that Norovirus and human Rotaviruses A were the main pathogens which caused viral diarrhea in Guangdong province. In summary, the multiplex rtPCR assay that could detect five viral pathogens in a single reaction was established. When performed in a 96-well real-time PCR system; more than 90 samples could be analyzed within three hours. The new method is simple, rapid, high sensitive and specific. The new assay can be applied to viral pathogens identification of viral diarrheal disease outbreak and provide the laboratory data for epidemiological research as well as clinical care of patients and then strengthen general public health. Acknowledgements This work was supported by National Natural Science Foundation of China (No. 81071433 to Q.H) and Shenzhen Public Service Platform of Pathogenic Microorganisms Repository. We thank Dr Zhaojun Duan from Chinese Center for Disease Control and Prevention for providing with virus positive samples. 7
136
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11
225 226
TABLE 1 Performance of Multiplex real time PCR assay compared to monoplex real time PCR No. of samples monoplex rtPCR Multiplex rtPCR Sapovirus Positive Negative Norovirus GI Positive Negative Rotavirus group A Positive Negative Adenovirus Positive Negative Norovirus GII Positive Negative Astrovirus Positive Negative
Sensitivity (%)
Specificity (%)
Agreement (%)
Kappa Value
0 808
100.0
100.0
100.0
1.00
6 2
0 804
75.0
100.0
99.7
0.85
116 10
5 681
92.0
99.2
98.2
0.92
22 1
1 788
95.7
99.9
99.8
0.98
190 12
6 604
94.0
99.0
97.8
0.94
8 1
1 802
88.9
99.9
99.8
0.88
Positive
Negative
4 0
227 228 229 230
12
231
FIGURE LEGENDS
232
Figure 1. Detection results of simulation for mixed infection according to CT value
233
Figure 1A and 1B indicate the mixed infection between Rotavirus group A (HRV) and Adenovirus
234
(AdV). Different copy numbers (0, 102, 104 and106 copies) of HRV were combined with 102
235
(series
236
duplicate experiments.
), 104 (series
) and106 (series
) copies of AdV. All CT values are means of
237
13
1
Simultaneous Detection of Five Enteric Viruses Associated with Gastroenteritis
2
Using a PCR Assay: a Single Real-time Multiplex Reaction and its Clinical Application
3 4
Yixiang Jiang1*, Lin Fang3*, Xiaolu Shi2*, Hailong Zhang2, Yinghui Li2, Yinman Lin2, Yaqun
5
Qiu2, Qingliang Chen4, Hui Li3, Li Zhou2†, Qinghua Hu1,2†
6 7
Affiliation:
8
1
9
2
School of Life Sciences, Shenzhen University, Shenzhen 518000, Guangdong, China Shenzhen Major Infectious Disease Control Key Laboratory, Shenzhen Center for Disease
10
Control and Prevention, Shenzhen 518055, Guangdong Province, China
11
3
12
Guangdong Province, China
13
4
14
*These authors contributed equally to this work.
15
† Correspondence authors:
16
L. Zhou, Shenzhen Centre for Disease Control and Prevention, Shenzhen 518055, Guangdong
17
Province, China. Phone: +86-755-25623237, Fax: +86-755-25623237.
18
E-mail: [email protected]
19
Q. Hu, Shenzhen Centre for Disease Control and Prevention, Shenzhen 518055, Guangdong
20
Province, China. Phone: +86-755-86580705, Fax: +86-755-25532595.
21
E-mail: [email protected]
Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 518000,
School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China
1
22
Running Title: Single reaction of five enteric viruses by multiplex real-time PCR
23
Key words: viral gastroenteritis, multiplex real-time PCR, the reverse transcription, single
24
Word count: Abstract 47, Text 1141
2
25
Abstract
26
We developed a highly sensitive, reverse transcription and multiplex real-time PCR (rtPCR)
27
assay that can identify five viruses; including six genogroups, in a single reaction: Norovirus
28
genogroups I, II;, Sapovirus genogroups I, II, IV and V, human Rotaviruses A, Adenovirus
29
serotypes 40 and 41, and human Astrovirus. In comparison to monoplex rtPCR assays,
30
sensitivities and specificities of the multiplex rtPCR ranged 75-100% and 99-100%, respectively,
31
when evaluated on 812 clinical stool specimens.
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 3
47
Norovirus genogroups I, II (NV) (1, 2), Sapovirus genogroups I, II, IV and V (SaV) (3),
48
human Rotaviruses A (HRV) (4), Adenovirus serotypes 40 and 41 (AdV) (5), and human
49
Astrovirus (AstV) (6) are the most common cause of viral gastroenteritis and result in large
50
outbreak of viral diarrhea (7-11). Rapid and accurate detection methods are essential for response
51
to viral diarrhea outbreaks and management of the patients (12, 13). Current detection methods
52
include electron microscopy (14), enzyme immunoassay (EIA) (15)，conventional or real-time
53
PCR (16-19), or the Luminex xMAP technology (12-13). These methods are laborious (14) or
54
need post gel electrophoresis (18) or allow for simultaneous detection of three or four viral
55
pathogens in one reaction (16,17,19), or need opening of reaction tubes for hybridization(12-13).
56
Here, based on the principle of multicolor combinational probe coding (MCPC) (20, 21), we
57
developed a more effective multiplex real-time PCR (rtPCR) assay for the simultaneous detection
58
of five major enteric viruses in a single reaction.
59
All gene sequences retrieved from GenBank were aligned using Clustal W software. The
60
primers and modified molecular beacon probes based on the conserved regions were designed
61
using Primer Premier (version 5.0), Oligo (version 6.31) and DNA folding software
62
(http://mfold.ma.albany.edu/?q=mfold/dna-folding-form). The sequences of the primers for
63
NV and HRV were adapted from the literature (19, 22). The primers had a common tag sequence
64
at the 5’ ends to keep the formation of primer dimmers to a minimum. Equine Arteritis virus
65
(EAV) was employed as an internal control to monitor the inhibition of amplification (23).
66 67 68
All primers and probes were commercially synthesized, labeled and purified by the company Sangon Biotech (Shanghai, China) (Supplementary Table S1). Ten positive samples in which SaV, NV GI, NV GII, HRV, AdV and AstV were identified by 4
69
PCR with sequencing were used for the plasmid DNA standards preparation and initial multiplex
70
rtPCR assay development. Viral DNA/RNA of clinical stool samples was extracted using the
71
Roche High Pure Vial Nucleic Acid kit (Roche Diagnostics, Penzberg, Germany). For the reverse
72
transcription, 5μl of extracted RNA was transcribed into cDNA using Primer Script 1st strand
73
cDNA Synthesis Kit (Takara Biotechnology Company, Dalian, China).
74
Multiplex rtPCR were conducted using a BIO-RAD CFX96 real-time PCR system. The final
75
volume was 25μl, which included 1×PCR buffer, 3mM MgCl2, 2μl of deoxynucleoside
76
triphosphate (2.5 mM), and 1 unit Taq polymerase, 0.1 μM to 0.9 μM specific primers and probes
77
(Supplementary Table S1) and 5μl of cDNA or DNA template. Optimal cycling conditions
78
included a first stage initiated by heating at 95°C for 3 min, followed by a second stage of five
79
cycles of 95 °C for 10 s, 58 °C for 20 s, 72 °C for 15s, and finally by a third stage of 40 cycles of
80
95 °C for 10s, 55 °C for 32s, 72 °C for 15s. FAM, HEX, ROX, Cy5 and qursar705 fluorescence was
81
detected and recorded at each step of primer annealing during the third stage.
82 83 84 85 86 87 88 89 90
To confirm whether the target genes selected were specific for each virus, nucleic acids of Giardia lamblia, Entamoeba histolytica, Cryptosporidium paHRVum, Staphylococcus aureus, Shigella sp., Yersinia enterocolitica, Campylobacter jejuni and Human enterovirus 71 and ten enteric viruses positive samples were tested by multiplex rtPCR. No cross-reaction was observed and all five of the targeted genes were identified correctly by the multiplex rtPCR (Supplementary Figure S1), indicating that the developed assay had good specificity. The detection limits of the multiplex rtPCR assays were assessed twice using seven serial ten-fold dilutions (107–101 copies/μl) of the plasmid DNA standards and four serial ten-fold dilutions (106-103 copies/μl) of the plasmid DNA standards were prepared for the analysis of the intro-reproducibility in triplicate 5
91 92 93 94 95
experiments. The detection limits of the five viruses were 10 copies / μl. Amplification efficiency ranged from 87.6% to 99.2% with strong correlation coefficients (R2≥0.99) (Supplementary Table S2 and Figure S2). The CT values from the triplicate experiments differ within one and the coefficient of variation (CV) for each target virus ranged from 0.06% to 2.3%, which indicated that the assay was reproducible (Supplementary Table S3).
96
Due to the clinical samples often containing mixed infections (24-27), HRV and AdV were
97
used as the representative in conjunction to simulate co-infection. Each of the 2.5μl plasmid DNA
98
standards were combined in equal amounts ranging from 102 to 106 copies / μl for multiplex rtPCR.
99
HRV and AdV could be simultaneously detected apart from when the number of HRV copies was
100
10,000 times higher than the number of AdV copies, and this prevented the AdV from being
101
detected (Figure 1).
102
To further validate the multiplex rtPCR assay’s specificity and sensitivity, 812 clinical stool
103
samples were collected from ten hospitals in four cities in Guangdong province and were detected
104
using the newly developed assay with direct comparison with commercial monoplex rtPCR kits
105
(ZJ Bio-Tech, Shanghai, China ). All monoplex rtPCR positive products of clinical samples were
106
sequenced for the further confirmation. Among the 812 stool samples, 372 were virus positive.
107
From which 29 stool specimens were found to be co-infected with two viruses, which included
108
11of NV GII and AdV, 10 of HRV and AdV, five of NV GII and HRV, as well as three of NV GI
109
and NV GII (Table 1).
110
When compared with monoplex rtPCR, the sensitivity of the multiplex rtPCR assay ranged
111
from 75% to100% and the specificity ranged from 99% to100%. The agreement ranged from
112
97.8%-100% and the kappa correlation ranged from 0.85 to 1.0 (Table 1). 6
113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129
130 131 132 133 134 135
This study has the limitation that requires further improvement. Two NV GI samples, 10 HRV samples, One AdV sample and 12 NV GII samples were positive by monoplex rtPCR but negative by multiplex rtPCR, which was due to nucleic acid degradation. More clinical samples will be collected for the further validation. Among 812 stool samples collected between January 2012 and July 2013, 422 stool samples were collected from Shenzhen, of which 216 were positive (including 116 NV II, 65 HRV, 17 AdV, eight NV I, eight AstV and two SaV); 222 from Guangzhou including 39 NV II, 55 HRV, four AdV, one AstV and two SaV positive; 108 from Dongguan with 40 NV II, three HRV and two AdV positive; and 60 from Zhongshan having seven NV II and three HRV positive (Supplementary Table S4). The data showed that Norovirus and human Rotaviruses A were the main pathogens which caused viral diarrhea in Guangdong province. In summary, the multiplex rtPCR assay that could detect five viral pathogens in a single reaction was established. When performed in a 96-well real-time PCR system; more than 90 samples could be analyzed within three hours. The new method is simple, rapid, high sensitive and specific. The new assay can be applied to viral pathogens identification of viral diarrheal disease outbreak and provide the laboratory data for epidemiological research as well as clinical care of patients and then strengthen general public health. Acknowledgements This work was supported by National Natural Science Foundation of China (No. 81071433 to Q.H) and Shenzhen Public Service Platform of Pathogenic Microorganisms Repository. We thank Dr Zhaojun Duan from Chinese Center for Disease Control and Prevention for providing with virus positive samples. 7
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TABLE 1 Performance of Multiplex real time PCR assay compared to monoplex real time PCR No. of samples monoplex rtPCR Multiplex rtPCR Sapovirus Positive Negative Norovirus GI Positive Negative Rotavirus group A Positive Negative Adenovirus Positive Negative Norovirus GII Positive Negative Astrovirus Positive Negative
Sensitivity (%)
Specificity (%)
Agreement (%)
Kappa Value
0 808
100.0
100.0
100.0
1.00
6 2
0 804
75.0
100.0
99.7
0.85
116 10
5 681
92.0
99.2
98.2
0.92
22 1
1 788
95.7
99.9
99.8
0.98
190 12
6 604
94.0
99.0
97.8
0.94
8 1
1 802
88.9
99.9
99.8
0.88
Positive
Negative
4 0
227 228 229 230
12
231
FIGURE LEGENDS
232
Figure 1. Detection results of simulation for mixed infection according to CT value
233
Figure 1A and 1B indicate the mixed infection between Rotavirus group A (HRV) and Adenovirus
234
(AdV). Different copy numbers (0, 102, 104 and106 copies) of HRV were combined with 102
235
(series
236
duplicate experiments.
), 104 (series
) and106 (series
) copies of AdV. All CT values are means of
237
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