Zbl. Bakt . 277, 84-89 (1992) © Gustav Fischer Verlag, SrungartlNew York
A Simplified Agarose Gel Electrophoresis for Rotavirus Detection REINHARD ZBINDEN , JOCHEN GOTTSCHALK , KONSTANZE MEZ, HEIDI NEUENSCHWANDER , and Iva HEINZER Institute of Microbiology, Kantonsspital Aarau, 5001 Aarau , Switzerland
With 3 Figures' Received July 18, 1991 . Revision received Octobe r 16, 1991 . Accepted January 3, 1992
Summary The use of agarose gel electrophoresis for rotav irus detection is a suitable routine method if simplifying modifications are introduced. Incorporation of ethidium bromide into the agarose gel permits its use for several times in the course of one week without further staining of the RNA segments. The electrophores is chamber with a LlV-Iight-transparenr bottom is put directly onto the transilluminator and the RNA bands of the rotavirus can be visualized without manipulating the gel tra y. During the winter of 1990/91 , we found a good sensitivity (98% ) of this method in comparison to a latex agglutination test (Slidex Rota-Kit 2®, bio Merieux),
Zusammenfassung Die Agarosegel-Elektrophorese fiir den Rotavirusnachweis ist nach wesentlichen Vereinfachungen - wie Vorfarbung des Agarosegels mit Ethidiumbromid und mehrfacher Verwendung des gleichen Gels - ein geeignetes Mittel fiir den Rotavirusnachweis in der Routinediagnostik. Die Uv-durchlassige Elektrophoresekammer mit dem Agarosegel wird auf den Transilluminator gestellt und die RNS-Banden der Rotaviren konnen direkt beobachtet werden. Wiihrend der Wintersaison 1990/91 zeigte diese Methode gegeniiber einem kommerziellen Latex-Agglutinationstest (Slidex Rota-Kit 2®, bio Merieux) eine gute Sensitivitat (98%).
Introduction Worldwide, rotaviru ses represent th e most frequent cause of acute gastroenteritis in childhood (2). Especiall y in children aged from 6 to 24 months, rotavirus infections pre sent them selves with symptoms of vomiting, diarrhea and fever (9). These manifes tat ion s mostly subside within one week, but in developing countries diarrhea is lifeth reatening. In temperate climates, rotaviru s infections show a peak incidence in winter
Rotavirus Detection in Agarose Gel
85
(6). Nosocomial infections are common in pediatric and nursery stations (5). Infection in neonates is accompanied by minor clinical symptoms or is indeed asymptomatic (3). Rotavirus infection is most simply diagnosed today with commercial enzyme immuno assays (EIA) or latex agglutination tests. Rotavirus capsid proteins are demonstrated directly in the feces. These tests show a good sensitivity and specificity in children with diarrhea. Especially in neonatal feces, ELISA tests occasionally show false-positive reactions (11). Polyacrylamide gel electrophoresis (PAGE) has become an important laboratory and epidemiological procedure for the characterization of rotaviral strains (7, 8, 10). The rotavirus genome consists of eleven double-stranded RNA segments, which can be separated in gel electrophoresis according to their molecular weight after extraction from the stool sample. Recently, agarose gel electrophoresis has been described as a screening test for the detection of rotavirus (1). We shall describe further modifications of this method and compare it with a latex agglutination test. The limitations of this agarose gel electrophoresis in comparison with PAGE are discussed.
Materials and Methods Patient material. Between November 1990 and March 1991 585 stool samples from children younger than 10 years were investigated for rotavirus by a latex agglutination test and agarose gel electrophoresis. In cases of discrepancies, both tests were repeated. The polyacrylamide gel electrophoresis (PAGE) was performed in some cases to detect different electrophorotypes. Latex agglutination test (Slidex Rota-Kit 2®, bio Merieux, 69280 Marcy-I'Etoile, France). Stool samples were suspended (10%) in 2 ml extraction buffer from the kit (not identical with the extraction buffer for the electrophoretic methods) and left at room temperature for 5 to 10 min. After centrifugation (800 g; 10 minutes), one drop of the supernatant was mixed with one drop of the sensitized latex reagent and examined for agglutination within 2 min; the negative control latex reagent was tested in parallel. Stool extraction for the electrophoretic methods. The extraction was performed as described by Chudzio et al. (1). Stool samples were suspended in 500 f.ll of extraction buffer (0.02 M Tris hydrochloride [pH 7.4], 0.3 M NaCl, 0.01 M MgCl z, 0.1 % sodium dodecyl sulphate, 0.005 M EDTA, 4% sucrose, 0.04% bromophenol blue) to a final concentration of 20%. Each preparation was mixed with an equal volume of phenol-chloroform (1: 1), vortexed for 30-45 s and centrifuged at 5500 g for 10 min. All manipulations with phenol should be carried out under a ventilated hood. The upper layer was transferred to another tube and stored at room temperature until 20 ul were loaded for the gel electrophoresis. Agarose gel electrophoresis. 3 g agarose (Standard low, BioRad) was suspended in 200 ml TBE buffer (0.09 M Tris borare, 0.002 M EDTA) and melted. Ethidium bromide (0.5 ug/ml) was added just before pouring the gel (0.5 ern/ 15 cm/25 ern) into a plastic tray. A comb was inserted to form loading wells. The solidified gel on the tray was submerged horizontally in TBE buffer and loaded with RNA extracts. The electrophoresis chamber with a UV transparent bottom (BioRad) was put onto the UV transilluminator and electrophoresis was carried out at 180 V for 1.5 h. Polyacrylamide gel electrophoresis (PAGE). For the vertical PAGE, discontinuous MiniProteanf II Gels (7.5%, BioRad) in the Mini-Protean'" II Dual Slab Cell (BioRad) were used. The electrode buffer (0.025 M Tris, 0.2 M glycin [pH 8.3]) was filled in to cover the gel. The wells were loaded and the electrophoresis was carried out at 75 V for 4 h. Afterwards, the gels were stained with the Silver Stain Kit (BioRad) and dried between two plastic foils in a Hoefer Easy Breezef drying frame (Hoefer Scientific Instruments, USA).
86
R. Zbinden et al. Results
The agar ose gel with incorporated ethid ium bromide could be used for several electrophoresis runs in the course of one week. Fig. 1 shows an agarose gel with the typical pattern of the RNA segments of rota virus-positive stool extracts from fWO subsequent days. The same lanes could be used several times, since the po sitive stool extracts disappeared tow ard s the anod e and new extracts could be loaded. In this way, we could test about 50 to 70 stoo l extra cts per gel. The segments could be demonstrated dur ing the electrophoresis run , since we put the electrophoresis chamber with a UV-transparent bottom onto the transillumiator. In the winter of 1990/91 , we tested all 585 samples with both methods. In comparison to the latex test, (Table 1) the agarose gel showed a good sensitivity (98%) .
Fig. 1. Lanes 1, 2, 5-9 show the typical pattern of ethidium bromide RNA segments of rotavirus-positive stool extracts (lane 7 is weakly positive). Lanes 5, 6 are from the previous day. Lanes 3 and 4 show negative extracts from two subsequent days with unspecific bands.
Rotavirus Detection in Agarose Gel
87
Table 1. Comparison of the agarose gel method and the latex test for rotavirus detection Latex test
+ Agarose gel electrophoresis
+
128
6
3
448
At the end of the year, a higher frequency of rotavirus infections was detected in two neighbouring pediatric stations within 5 days. Fig. 2 shows a series of rotavirus-positive stool samples which had the same pattern in the agarose gel electrophoresis, because segments 7,8,9 were not separated with this method. PAGE (Fig. 3), however, showed different patterns, i.e. different electrophorotypes. In lanes 1 and 2, 4 to 6 and 8 segments 7 and 8 comigrated, but in lane 7, segments 7, 8, 9 could be distinguished; in lane 3, the distinction of segments 7, 8, 9 was only clearly visible on the fixed and dried gel, but not on Fig. 3 (photograph). Lanes 3 and 7 were clearly different from the identical patterns of the other 6 cases.
Fig. 2. A series of rotavirus-positive stool samples with the same pattern in agarose gel electrophoresis. Segments 7, 8, 9 are not separated.
88
R. Zbinden et al.
-
Fig. 3. The same series of rotavirus-positive stool samples as in Fig. 2 in polyacrylamide gel electrophoresis with different electrophoro types. In lane 7, the segments 7, 8, 9 can be distinguished; in lane 3, the separation of segments 7, 8, 9 is only visible on the dried gel. In the other lanes, segments 7, 8 comigrate (see text ).
Discussion Agarose gel electro phoresis is a good alternative to other tests for detecting rot avirus. Especially the simplificatio ns made it possible to introduce this technique into routine diagnosis. The pre-stained gel can be used several times for one week and a special wast e disposal of staining solutions as in silverstaining is not necessary. The gel lanes can be loaded several times as the RNA segments of positive stool extracts move towa rds the anode and do not influence the identification of the next positive stool ext racts. The electro pho resis cha mber with the UV-light-tran spa rent bottom on the transillumin ato r allows th e direct illustration of the segments during the segregation. After 10 min, the cover of the electrophoresis chamber is moistened by buffer evaporation. In this case, one sho uld wait until segregation is finished or stop the electro phoresis to clean the cover. At the end of one week, the inten sity of the stained segments of the rotavirus is still intense enough. In doubtful cases, the supp ort tray with the gel can be tak en out of the electrophoresis chamber and put directly onto the transilluminator so that the bands can be seen more clearly. Weakl y positive result s as show n in Fig. 1 (lane 7) are commonly ob served in less than 10% of cases. With this method we did not have false positive readin gs, as the pattern of the RNA segments has been typical of the rotavirus, and in comparison to the latex test, the sensitivity (98 % ) has been very good in our survey. Reading errors of the latex test (nearly 3 %, data not shown) wer e especially produced by frequ ent shifting of the technicians within the laboratory and were detected on the basis of discrepancies revealed by agarose gel electrophoresis; on repeating concordant results were found. In compa rison to electro n microscopy (4), the Slidex Rota-Kit 2 using a monoclonal ant ibody showe d a sensitivity of 96 % and a specificity of 100%.
Rotavirus Detection in Agarose Gel
89
Th e initial cost of equipment for the agarose gel electrophoresis is high, but such an electrophoresis chamber and the power supply today belong to the sta nda rd equipment of a microbi ological lab or ator y. Th e cost per test is mu ch higher for the latex test than it is for the agarose gel electroph oresis which is easy to perform since staining procedures are not needed . The latex test is not as easy to perform as it seems, especiall y when the technician s are not used to it. Th e extraction time for the stoo l samples is nearl y the same for both meth od s. So the tot al time required to report the agarose gel electrophore sis result s is two hours. For epidemiological examin at ion s, this method is not sufficient, as bands 7, 8, 9 are not separa ted; in the event of suspected identity, PAGE can be initiated with the same stool extrac t. In conclusion, the simplified agarose gel electro phoresis has been found to be a suita ble test for rotaviru s detection .
References 1. Chudz io, T., S. Kasatiya , N. Irvine, and P. Sankar-Mistry: Rapid Screening test for the
diagnosis of rotaviru s infection. ] . Clin. Microbiol. 27 (1989) 2394-2396 2. Estes, M. K., E. L. Palmer, and j. F. Obijesk i: Rotaviruses: a review. In: Current Topics
in Microbiology and Immunology 105, pp. 124-1 84. Springer-Verlag, Berlin-Heidelberg-New York-Tokyo (1983) 3. Kunz, j. , R. Slongo, M. Schams, and R. Zbinden : An outbreak of rotavirus infecrions in newborns - New aspects? J. Perinat, Med. 18 (1990) 357-362 4. Martin, A . L., P. j. l\lfolyne
A Simplified Agarose Gel Electrophoresis for Rotavirus Detection REINHARD ZBINDEN , JOCHEN GOTTSCHALK , KONSTANZE MEZ, HEIDI NEUENSCHWANDER , and Iva HEINZER Institute of Microbiology, Kantonsspital Aarau, 5001 Aarau , Switzerland
With 3 Figures' Received July 18, 1991 . Revision received Octobe r 16, 1991 . Accepted January 3, 1992
Summary The use of agarose gel electrophoresis for rotav irus detection is a suitable routine method if simplifying modifications are introduced. Incorporation of ethidium bromide into the agarose gel permits its use for several times in the course of one week without further staining of the RNA segments. The electrophores is chamber with a LlV-Iight-transparenr bottom is put directly onto the transilluminator and the RNA bands of the rotavirus can be visualized without manipulating the gel tra y. During the winter of 1990/91 , we found a good sensitivity (98% ) of this method in comparison to a latex agglutination test (Slidex Rota-Kit 2®, bio Merieux),
Zusammenfassung Die Agarosegel-Elektrophorese fiir den Rotavirusnachweis ist nach wesentlichen Vereinfachungen - wie Vorfarbung des Agarosegels mit Ethidiumbromid und mehrfacher Verwendung des gleichen Gels - ein geeignetes Mittel fiir den Rotavirusnachweis in der Routinediagnostik. Die Uv-durchlassige Elektrophoresekammer mit dem Agarosegel wird auf den Transilluminator gestellt und die RNS-Banden der Rotaviren konnen direkt beobachtet werden. Wiihrend der Wintersaison 1990/91 zeigte diese Methode gegeniiber einem kommerziellen Latex-Agglutinationstest (Slidex Rota-Kit 2®, bio Merieux) eine gute Sensitivitat (98%).
Introduction Worldwide, rotaviru ses represent th e most frequent cause of acute gastroenteritis in childhood (2). Especiall y in children aged from 6 to 24 months, rotavirus infections pre sent them selves with symptoms of vomiting, diarrhea and fever (9). These manifes tat ion s mostly subside within one week, but in developing countries diarrhea is lifeth reatening. In temperate climates, rotaviru s infections show a peak incidence in winter
Rotavirus Detection in Agarose Gel
85
(6). Nosocomial infections are common in pediatric and nursery stations (5). Infection in neonates is accompanied by minor clinical symptoms or is indeed asymptomatic (3). Rotavirus infection is most simply diagnosed today with commercial enzyme immuno assays (EIA) or latex agglutination tests. Rotavirus capsid proteins are demonstrated directly in the feces. These tests show a good sensitivity and specificity in children with diarrhea. Especially in neonatal feces, ELISA tests occasionally show false-positive reactions (11). Polyacrylamide gel electrophoresis (PAGE) has become an important laboratory and epidemiological procedure for the characterization of rotaviral strains (7, 8, 10). The rotavirus genome consists of eleven double-stranded RNA segments, which can be separated in gel electrophoresis according to their molecular weight after extraction from the stool sample. Recently, agarose gel electrophoresis has been described as a screening test for the detection of rotavirus (1). We shall describe further modifications of this method and compare it with a latex agglutination test. The limitations of this agarose gel electrophoresis in comparison with PAGE are discussed.
Materials and Methods Patient material. Between November 1990 and March 1991 585 stool samples from children younger than 10 years were investigated for rotavirus by a latex agglutination test and agarose gel electrophoresis. In cases of discrepancies, both tests were repeated. The polyacrylamide gel electrophoresis (PAGE) was performed in some cases to detect different electrophorotypes. Latex agglutination test (Slidex Rota-Kit 2®, bio Merieux, 69280 Marcy-I'Etoile, France). Stool samples were suspended (10%) in 2 ml extraction buffer from the kit (not identical with the extraction buffer for the electrophoretic methods) and left at room temperature for 5 to 10 min. After centrifugation (800 g; 10 minutes), one drop of the supernatant was mixed with one drop of the sensitized latex reagent and examined for agglutination within 2 min; the negative control latex reagent was tested in parallel. Stool extraction for the electrophoretic methods. The extraction was performed as described by Chudzio et al. (1). Stool samples were suspended in 500 f.ll of extraction buffer (0.02 M Tris hydrochloride [pH 7.4], 0.3 M NaCl, 0.01 M MgCl z, 0.1 % sodium dodecyl sulphate, 0.005 M EDTA, 4% sucrose, 0.04% bromophenol blue) to a final concentration of 20%. Each preparation was mixed with an equal volume of phenol-chloroform (1: 1), vortexed for 30-45 s and centrifuged at 5500 g for 10 min. All manipulations with phenol should be carried out under a ventilated hood. The upper layer was transferred to another tube and stored at room temperature until 20 ul were loaded for the gel electrophoresis. Agarose gel electrophoresis. 3 g agarose (Standard low, BioRad) was suspended in 200 ml TBE buffer (0.09 M Tris borare, 0.002 M EDTA) and melted. Ethidium bromide (0.5 ug/ml) was added just before pouring the gel (0.5 ern/ 15 cm/25 ern) into a plastic tray. A comb was inserted to form loading wells. The solidified gel on the tray was submerged horizontally in TBE buffer and loaded with RNA extracts. The electrophoresis chamber with a UV transparent bottom (BioRad) was put onto the UV transilluminator and electrophoresis was carried out at 180 V for 1.5 h. Polyacrylamide gel electrophoresis (PAGE). For the vertical PAGE, discontinuous MiniProteanf II Gels (7.5%, BioRad) in the Mini-Protean'" II Dual Slab Cell (BioRad) were used. The electrode buffer (0.025 M Tris, 0.2 M glycin [pH 8.3]) was filled in to cover the gel. The wells were loaded and the electrophoresis was carried out at 75 V for 4 h. Afterwards, the gels were stained with the Silver Stain Kit (BioRad) and dried between two plastic foils in a Hoefer Easy Breezef drying frame (Hoefer Scientific Instruments, USA).
86
R. Zbinden et al. Results
The agar ose gel with incorporated ethid ium bromide could be used for several electrophoresis runs in the course of one week. Fig. 1 shows an agarose gel with the typical pattern of the RNA segments of rota virus-positive stool extracts from fWO subsequent days. The same lanes could be used several times, since the po sitive stool extracts disappeared tow ard s the anod e and new extracts could be loaded. In this way, we could test about 50 to 70 stoo l extra cts per gel. The segments could be demonstrated dur ing the electrophoresis run , since we put the electrophoresis chamber with a UV-transparent bottom onto the transillumiator. In the winter of 1990/91 , we tested all 585 samples with both methods. In comparison to the latex test, (Table 1) the agarose gel showed a good sensitivity (98%) .
Fig. 1. Lanes 1, 2, 5-9 show the typical pattern of ethidium bromide RNA segments of rotavirus-positive stool extracts (lane 7 is weakly positive). Lanes 5, 6 are from the previous day. Lanes 3 and 4 show negative extracts from two subsequent days with unspecific bands.
Rotavirus Detection in Agarose Gel
87
Table 1. Comparison of the agarose gel method and the latex test for rotavirus detection Latex test
+ Agarose gel electrophoresis
+
128
6
3
448
At the end of the year, a higher frequency of rotavirus infections was detected in two neighbouring pediatric stations within 5 days. Fig. 2 shows a series of rotavirus-positive stool samples which had the same pattern in the agarose gel electrophoresis, because segments 7,8,9 were not separated with this method. PAGE (Fig. 3), however, showed different patterns, i.e. different electrophorotypes. In lanes 1 and 2, 4 to 6 and 8 segments 7 and 8 comigrated, but in lane 7, segments 7, 8, 9 could be distinguished; in lane 3, the distinction of segments 7, 8, 9 was only clearly visible on the fixed and dried gel, but not on Fig. 3 (photograph). Lanes 3 and 7 were clearly different from the identical patterns of the other 6 cases.
Fig. 2. A series of rotavirus-positive stool samples with the same pattern in agarose gel electrophoresis. Segments 7, 8, 9 are not separated.
88
R. Zbinden et al.
-
Fig. 3. The same series of rotavirus-positive stool samples as in Fig. 2 in polyacrylamide gel electrophoresis with different electrophoro types. In lane 7, the segments 7, 8, 9 can be distinguished; in lane 3, the separation of segments 7, 8, 9 is only visible on the dried gel. In the other lanes, segments 7, 8 comigrate (see text ).
Discussion Agarose gel electro phoresis is a good alternative to other tests for detecting rot avirus. Especially the simplificatio ns made it possible to introduce this technique into routine diagnosis. The pre-stained gel can be used several times for one week and a special wast e disposal of staining solutions as in silverstaining is not necessary. The gel lanes can be loaded several times as the RNA segments of positive stool extracts move towa rds the anode and do not influence the identification of the next positive stool ext racts. The electro pho resis cha mber with the UV-light-tran spa rent bottom on the transillumin ato r allows th e direct illustration of the segments during the segregation. After 10 min, the cover of the electrophoresis chamber is moistened by buffer evaporation. In this case, one sho uld wait until segregation is finished or stop the electro phoresis to clean the cover. At the end of one week, the inten sity of the stained segments of the rotavirus is still intense enough. In doubtful cases, the supp ort tray with the gel can be tak en out of the electrophoresis chamber and put directly onto the transilluminator so that the bands can be seen more clearly. Weakl y positive result s as show n in Fig. 1 (lane 7) are commonly ob served in less than 10% of cases. With this method we did not have false positive readin gs, as the pattern of the RNA segments has been typical of the rotavirus, and in comparison to the latex test, the sensitivity (98 % ) has been very good in our survey. Reading errors of the latex test (nearly 3 %, data not shown) wer e especially produced by frequ ent shifting of the technicians within the laboratory and were detected on the basis of discrepancies revealed by agarose gel electrophoresis; on repeating concordant results were found. In compa rison to electro n microscopy (4), the Slidex Rota-Kit 2 using a monoclonal ant ibody showe d a sensitivity of 96 % and a specificity of 100%.
Rotavirus Detection in Agarose Gel
89
Th e initial cost of equipment for the agarose gel electrophoresis is high, but such an electrophoresis chamber and the power supply today belong to the sta nda rd equipment of a microbi ological lab or ator y. Th e cost per test is mu ch higher for the latex test than it is for the agarose gel electroph oresis which is easy to perform since staining procedures are not needed . The latex test is not as easy to perform as it seems, especiall y when the technician s are not used to it. Th e extraction time for the stoo l samples is nearl y the same for both meth od s. So the tot al time required to report the agarose gel electrophore sis result s is two hours. For epidemiological examin at ion s, this method is not sufficient, as bands 7, 8, 9 are not separa ted; in the event of suspected identity, PAGE can be initiated with the same stool extrac t. In conclusion, the simplified agarose gel electro phoresis has been found to be a suita ble test for rotaviru s detection .
References 1. Chudz io, T., S. Kasatiya , N. Irvine, and P. Sankar-Mistry: Rapid Screening test for the
diagnosis of rotaviru s infection. ] . Clin. Microbiol. 27 (1989) 2394-2396 2. Estes, M. K., E. L. Palmer, and j. F. Obijesk i: Rotaviruses: a review. In: Current Topics
in Microbiology and Immunology 105, pp. 124-1 84. Springer-Verlag, Berlin-Heidelberg-New York-Tokyo (1983) 3. Kunz, j. , R. Slongo, M. Schams, and R. Zbinden : An outbreak of rotavirus infecrions in newborns - New aspects? J. Perinat, Med. 18 (1990) 357-362 4. Martin, A . L., P. j. l\lfolyne
