Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses

被引:46
作者
Linssen, B
Kinney, RM
Aguilar, P
Russell, KL
Watts, DM
Kaaden, OR
Pfeffer, M
机构
[1] Univ Munich, Inst Med Microbiol Infect & Epidem Dis, D-80539 Munich, Germany
[2] Ctr Dis Control & Prevent, Div Vector Borne Infect Dis, Natl Ctr Infect Dis, Publ Hlth Serv,US Dept Hlth & Serv, Ft Collins, CO USA
[3] NAMRID, Naval Med Res Ctr Detachment, Unit 3800, APO, AA 34031 USA
关键词
D O I
10.1128/JCM.38.4.1527-1535.2000
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species, The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and rv), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes WE, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype LAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR, The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos, All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.
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页码:1527 / 1535
页数:9
相关论文
共 51 条
[1]   SENSITIVE AND SPECIFIC COLORIMETRIC DOT ASSAY TO DETECT EASTERN EQUINE ENCEPHALOMYELITIS VIRAL-RNA IN MOSQUITOS (DIPTERA, CULICIDAE) AFTER POLYMERASE CHAIN-REACTION AMPLIFICATION [J].
ARMSTRONG, P ;
BOROVSKY, D ;
SHOPE, RE ;
MORRIS, CD ;
MITCHELL, CJ ;
KARABATSOS, N ;
KOMAR, N ;
SPIELMAN, A .
JOURNAL OF MEDICAL ENTOMOLOGY, 1995, 32 (01) :42-52
[2]   Genetic targets for the detection and identification of Venezuelan equine encephalitis viruses [J].
Brightwell, G ;
Brown, JM ;
Coates, DM .
ARCHIVES OF VIROLOGY, 1998, 143 (04) :731-742
[3]   IDENTIFICATION OF A NEW VENEZUELAN EQUINE ENCEPHALITIS-VIRUS FROM BRAZIL [J].
CALISHER, CH ;
KINNEY, RM ;
LOPES, OD ;
TRENT, DW ;
MONATH, TP ;
FRANCY, DB .
AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 1982, 31 (06) :1260-1272
[4]   REEVALUATION OF THE WESTERN EQUINE ENCEPHALITIS ANTIGENIC COMPLEX OF ALPHAVIRUSES (FAMILY TOGAVIRIDAE) AS DETERMINED BY NEUTRALIZATION TESTS [J].
CALISHER, CH ;
KARABATSOS, N ;
LAZUICK, JS ;
MONATH, TP ;
WOLFF, KL .
AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 1988, 38 (02) :447-452
[5]   ARBOVIRUS INVESTIGATIONS IN ARGENTINA, 1977-1980 .3. IDENTIFICATION AND CHARACTERIZATION OF VIRUSES ISOLATED, INCLUDING NEW SUBTYPES OF WESTERN AND VENEZUELAN EQUINE ENCEPHALITIS VIRUSES AND 4 NEW BUNYAVIRUSES (LAS MALOYAS, RESISTENCIA, BARRANQUERAS, AND ANTEQUERA) [J].
CALISHER, CH ;
MONATH, TP ;
MITCHELL, CJ ;
SABATTINI, MS ;
CROPP, CB ;
KERSCHNER, J ;
HUNT, AR ;
LAZUICK, JS .
AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 1985, 34 (05) :956-965
[6]   VENEZUELAN EQUINE ENCEPHALITIS VIRUS FROM SOUTH FLORIDA [J].
CHAMBERLAIN, RW ;
COLEMAN, PH ;
SUDIA, WD ;
WORK, TH .
SCIENCE, 1964, 145 (362) :272-&
[7]   NUCLEOTIDE-SEQUENCE OF THE GENOME REGION ENCODING THE 26S-MESSENGER RNA OF EASTERN EQUINE ENCEPHALOMYELITIS VIRUS AND THE DEDUCED AMINO-ACID-SEQUENCE OF THE VIRAL STRUCTURAL PROTEINS [J].
CHANG, GJJ ;
TRENT, DW .
JOURNAL OF GENERAL VIROLOGY, 1987, 68 :2129-2142
[8]   GENOME SEQUENCES OF A MOUSE-AVIRULENT AND A MOUSE-VIRULENT STRAIN OF ROSS RIVER VIRUS [J].
FARAGHER, SG ;
MEEK, ADJ ;
RICE, CM ;
DALGARNO, L .
VIROLOGY, 1988, 163 (02) :509-526
[9]   BIOCHEMICAL AND ANTIGENIC COMPARISONS OF THE ENVELOPE GLYCOPROTEINS OF VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS-STRAINS [J].
FRANCE, JK ;
WYRICK, BC ;
TRENT, DW .
JOURNAL OF GENERAL VIROLOGY, 1979, 44 (SEP) :725-740
[10]   NUCLEOTIDE-SEQUENCE OF CDNA CODING FOR SEMLIKI-FOREST VIRUS MEMBRANE-GLYCOPROTEINS [J].
GAROFF, H ;
FRISCHAUF, AM ;
SIMONS, K ;
LEHRACH, H ;
DELIUS, H .
NATURE, 1980, 288 (5788) :236-241