Genogroup I and II Noroviruses Detected in Stool Samples by Real-Time Reverse Transcription-PCR Using Highly Degenerate Universal Primers

Gary P. Richards; Michael A. Watson; Rebecca L. Fankhauser; Stephan S. Monroe

doi:10.1128/AEM.70.12.7179-7184.2004

DOI: 10.1128/AEM.70.12.7179-7184.2004

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FIG. 1.
Results of real-time RT-PCR amplification of genogroup I (A and B) and II (C to F) noroviruses and NC (F) from stool extracts. Optics graphs (A, C, and E) show amplicon production, and first-derivative melt graphs (B, D, and F) show corresponding melting temperatures for each of 13 norovirus clusters.
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FIG. 2.
Comparison of (A) optics graphs, (B) first-derivative melt curves, (C) electrophoretic gel, and (D) Ct values for low-titer norovirus RNA amplification by real-time RT-PCR with optical reads performed at 60 and 77°C. (C) Electrophoretic gel with 100-bp ladder (Mr). Lanes: 1, amplicon produced with an optical read at 60°C; 2, amplicon with an optical read at 77°C; 3 and 4, negative controls with optical reads performed at 60 and 77°C, respectively. The 213-bp amplicon represents authentic norovirus amplicon, and the lower-mass products represent primer dimers. The optical read temperatures and corresponding Ct values derived from panel A are shown in the table in panel D.
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FIG. 3.
Detection of two noroviruses in stool samples by first-derivative melt curves. GI/1 and GII/1 noroviruses, representing Norwalk and Hawaii viruses, respectively, are simultaneously detected in a single stool extract.

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TABLE 1.

Primer sets evaluated for real-time RT-PCR

Norovirus genogroup	Primer designation	Sequence (description)^a	Amplicon size (bp)
I	MON 432	5′ TGG ACI CGY GGI CCY AAY CA 3′ (RNA sense)
	MON 434	5′ GAA SCG CAT CCA RCG GAA CAT 3′ (cDNA sense)	213
II	MON 431	5′ TGG ACI AGR GGI CCY AAY CA 3′ (RNA sense)
	MON 433	5′ GGA YCT CAT CCA YCT GAA CAT 3′ (cDNA sense)	213

↵ a Ambiguity code: I, inosine; R, purine (A/G); Y, pyrimidine (C/T); S, strong (C/G).

TABLE 2.

Noroviruses amplified by real-time RT-PCR of stool extracts and accompanying T_ms of the amplicons, slopes of the standard curves, correlation coefficients (R²) of the standard curves, and the presence of inhibitors in 1 μl of undiluted extract

Genogroup	Cluster^a	Representative virus	T_m (°C)	Slope	R²	Inhibitors^b
I	1	Norwalk	81.84	−0.344	0.999	No
	2	Southampton	83.00	−0.447	0.997	No
	3	Desert Shield	82.39	−0.357	0.984	Yes
	4	Chiba	83.79	−0.803	0.974	No
	5	Musgrove	83.17	−0.388	0.981	No
II	1	Hawaii	83.82	−0.390	0.970	Yes
	2	Snow Mountain	82.67	−0.428	0.991	No
	3	Toronto	81.18	−0.459	1.000	No
	4	Lordsdale	83.96	−0.400	0.968	Yes
	5	Hillingdon	83.34	−0.364	0.998	Yes
	6	Seacroft	84.90	−0.669	1.000	Yes
	7	Leeds	84.90	−0.590	1.000	Yes
	8	Amsterdam	82.81	−0.337	0.995	No

↵ a Genetic clusters are based on criteria described by Ando et al. (1).
↵ b A “yes” indicates that the real-time RT-PCR reaction was partially inhibited by the use of 1 μl of stool extract in the real-time RT-PCR reaction mix.

TABLE 3.

Ct values and T_ms for noroviruses detected by real-time RT-PCR of stool samples obtained from health department laboratories

Sample origin	Ct	T_m (°C)
Delaware	21.99	84.04
	26.10	83.88
	24.17	83.34
	26.23	83.94
	21.36	83.99
	26.09	83.90
	21.66	83.90
	20.14	83.67
	26.11	83.81
	21.46	84.21
	23.33	83.92
	18.10	83.80
	25.83	84.08
	22.16	83.60
Florida	21.72	83.00
Rhode Island	22.74	85.12
	18.13	84.13
	26.42	83.09
	25.58	83.23
	20.44	82.96
	23.73	83.18
	17.42	83.38
	20.26	83.54
	16.62	83.35