(~) INSTITUT PASTEUR/ELSEVIER Paris 1990
Res. 'Y"I-! -0 1 '. 1990, 141, 455-457
BRIEF NOTE
USE OF DNA AMPLIFICATION FOR RAPID
DETECTION OF DENGUE VIRUSES IN MIDGUT CELLS OF INDIVIDUAL MOSQUITOES I. Tardieux and O. Poupei Unitd d'Ecologie des systbmes vectoriels, Institut Pasteur, 75724 Paris Cedex 15
The detection of arthropod-borne pathogens in their vector species is essential both for the study of fundamental vector-pathogen relationships and in order to plan and monitor vector control programmes. For Flaviviruses, especially dengue viruses, the method used to date for recognizing infected mosquitoes is based on direct antigen detection in brain tissue squashes containing large amounts of viral material, using an immunofluorescence technique (Kuberski and Rosen, 1977). This method is both time-consuming and of limited sensitivity. In contrast, the method we describe here enables the detection of only a few viral particles in the midgut cells of an individual mosquito, containing about 103 cells. Very early after experimental infectious blood feeding of the mosquito (Aedes albopictus), we used a polymerase chain reaction (PCR) to amplify a specific 289-bp fragment of the envelope gene from an RNA-cDNA hybrid. Each mosquito was dissected in STE buffer pH 8 (100 mM NaC1, 10 mM Tris-C1 pH 8, 1 mM EDTA pH 8) precleaned with 0.2 °70 DEPC (diethylpyrocarbonate, an inhibitor of RNase). The mesenteron was removed, washed three times in STE and dilacerated in 100 ~1 of 0.1 x STE, 0.5 070 NP40 for 5 rain on ice. Then, 20 units of RNase inhibkor and 2 ~g of proteinase K were added before a 30-min incubation at 56°C. The suspension was spun for 10 min at 4°C and 2,000 rpm. RNA was ethanol-precipitated from the phenol-extracted supernatant with acrylamide
Submitted March 22, 1990, accepted April 20, 1990.
456
L T A R D I E U X A N D O. P O U P E L
carrier and resuspended in 20 ~1 of sterile water. The first strand cDNA was synthetized using a 19-bp-specific oligomer: in a final volume of 20 l.d 1 × RT buffer (140 mM KCI, 100 mM Tris-Cl pH 8.3, 20 mM MgC12, 20 mM DTT) mixed with 20 units of RNase inhibitor, 0.25 mM of each dNTP, 0.02 mM of primer, 10 t-d of RNA template and 10 units of AMV-RT. After a 30-min incubation at 42°C and ethanol precipitation, the RNAcDNA complex was suspended in 50 t~l of sterile water. For the PCR, 60 nM of each dNTP, 10 nM of the first primer used for cDNA and 10 nM of another 20-bp-specific oligomer, 10 vd of RNA-cDNA complex and 1.25 units of TAQDNA polymerase were mixed in a final volume of 100 ~l of I x TAQ buffer Cetus, and submitted *.o 40 cycles in a "Perkip Elmer" apparatus as follows: 30 s for denaturation at 94°C, 1 min for annealing at 58°C and 1 min for elongation at 72°C (Saiki et al., 1988).
E
FG
"A B
C D E F
FIG. 1 (left). - - 1.2 % Agarose gel electrophoresis of PCR products. A) Supernatant-infected C6-36 cell culture, 6 days after infection. B) A mosquito (A. albopictus, Culicidae), fed on an infectious blood meal, 3 days prior to dissection. C) A control from the supernatant of the last midgut wash. D) and E) A mosquito fed on a blood meal containing respectively, no virus and heated virus (1 h at 65°C). F) Control without RNA. G) ~X174 DNA HaelII digest. FIG. 2 (right). - - Southern blotting of the gel from figure 1. Hybridization of a 20-bp oligomer, using washes in 6 x NaCl/citrate or 3.0 M Me4NCI.
AMV-RT = avian myeloblastosis virus/reverse transcriptase.
I
I
PCR = polymerase chain reaction.
PCR D E T E C T I O N OF D E N G U E VIRUS I N M O S Q U I T O
457
As seen in lane B of the 1.2 % agarose gel (fig. 1), PCR amplification of RNA from infected-mosquito midgut revealed a 289-bp band, the specificity of which was confirmed by Southern blotting and hybridization with a 20-bp oligomer which did not overlap those used for amplification (fig. 2), according to standard protocols (Wallace et al., 1981 ; Wood et aL, 1985). Thus, PCR amplification of material obtained from infected-mosquito midgut provided a straightforward and sensitive method of detection of dengue virus infection in a given tissue of a single mosquito. KEY-WORDS: Aedes albopictus, Dengue; PCR, Note.
RI~SUME UT!LISAT!ON DE L'AMPLIFICATIONGI~NIQUEPOUR LA DI~TECTIONRAPIDE DE PART1CULESVIRALESCHEZ LE MOUSTIQUE
Nous pr6sentons un protocole de PCR (amplification enzymatique g6nique), permettant la d~tection de particules virales de dengue au sein de cellules de l'6pith61ium intestinal du moustique vecteur, Aedes albopictus, apr6s infection par voie orale. MOTS-CLI~S: Aedes albopictus, Dengue; PCR; Note.
REFERENCES KUBERSKI, T.T. & ROSEN, L. (1977), A simple technique for the detection of dengue
antigen in mosquitoes by immunofluorescence. Amer. J. trop. Med. Hyg., 26, 533-537. SAIKi, R.K., GELFANO,D.H., STOFFEL,S., SCHARF, S.J., HIGUSHI, R., HORN, G.T., MULLIS, K.B. & ERLICH, H.A. (1988), Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491. WALLACE, R.B., JOHNSON, M.J., HIROSE, T., MIVAKE, T., KAWASHIMA, E.H. & ITAKURA,K. (1981), The use of synthetic oligonucleotides as hybridization probes. -- II. Hybridization of oligonucleotides of mixed sequence to rabbit B-globin DNA. Nucl. Acids Res., 9, 879-894. WOOD, W.I., GITSCHIER,J., LASKY,L.A. & LAWN, R.M. (1985), Base composkionindependent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc. nat. Acad. Sci. (Wash.), 82, 1585-1588.
Res. 'Y"I-! -0 1 '. 1990, 141, 455-457
BRIEF NOTE
USE OF DNA AMPLIFICATION FOR RAPID
DETECTION OF DENGUE VIRUSES IN MIDGUT CELLS OF INDIVIDUAL MOSQUITOES I. Tardieux and O. Poupei Unitd d'Ecologie des systbmes vectoriels, Institut Pasteur, 75724 Paris Cedex 15
The detection of arthropod-borne pathogens in their vector species is essential both for the study of fundamental vector-pathogen relationships and in order to plan and monitor vector control programmes. For Flaviviruses, especially dengue viruses, the method used to date for recognizing infected mosquitoes is based on direct antigen detection in brain tissue squashes containing large amounts of viral material, using an immunofluorescence technique (Kuberski and Rosen, 1977). This method is both time-consuming and of limited sensitivity. In contrast, the method we describe here enables the detection of only a few viral particles in the midgut cells of an individual mosquito, containing about 103 cells. Very early after experimental infectious blood feeding of the mosquito (Aedes albopictus), we used a polymerase chain reaction (PCR) to amplify a specific 289-bp fragment of the envelope gene from an RNA-cDNA hybrid. Each mosquito was dissected in STE buffer pH 8 (100 mM NaC1, 10 mM Tris-C1 pH 8, 1 mM EDTA pH 8) precleaned with 0.2 °70 DEPC (diethylpyrocarbonate, an inhibitor of RNase). The mesenteron was removed, washed three times in STE and dilacerated in 100 ~1 of 0.1 x STE, 0.5 070 NP40 for 5 rain on ice. Then, 20 units of RNase inhibkor and 2 ~g of proteinase K were added before a 30-min incubation at 56°C. The suspension was spun for 10 min at 4°C and 2,000 rpm. RNA was ethanol-precipitated from the phenol-extracted supernatant with acrylamide
Submitted March 22, 1990, accepted April 20, 1990.
456
L T A R D I E U X A N D O. P O U P E L
carrier and resuspended in 20 ~1 of sterile water. The first strand cDNA was synthetized using a 19-bp-specific oligomer: in a final volume of 20 l.d 1 × RT buffer (140 mM KCI, 100 mM Tris-Cl pH 8.3, 20 mM MgC12, 20 mM DTT) mixed with 20 units of RNase inhibitor, 0.25 mM of each dNTP, 0.02 mM of primer, 10 t-d of RNA template and 10 units of AMV-RT. After a 30-min incubation at 42°C and ethanol precipitation, the RNAcDNA complex was suspended in 50 t~l of sterile water. For the PCR, 60 nM of each dNTP, 10 nM of the first primer used for cDNA and 10 nM of another 20-bp-specific oligomer, 10 vd of RNA-cDNA complex and 1.25 units of TAQDNA polymerase were mixed in a final volume of 100 ~l of I x TAQ buffer Cetus, and submitted *.o 40 cycles in a "Perkip Elmer" apparatus as follows: 30 s for denaturation at 94°C, 1 min for annealing at 58°C and 1 min for elongation at 72°C (Saiki et al., 1988).
E
FG
"A B
C D E F
FIG. 1 (left). - - 1.2 % Agarose gel electrophoresis of PCR products. A) Supernatant-infected C6-36 cell culture, 6 days after infection. B) A mosquito (A. albopictus, Culicidae), fed on an infectious blood meal, 3 days prior to dissection. C) A control from the supernatant of the last midgut wash. D) and E) A mosquito fed on a blood meal containing respectively, no virus and heated virus (1 h at 65°C). F) Control without RNA. G) ~X174 DNA HaelII digest. FIG. 2 (right). - - Southern blotting of the gel from figure 1. Hybridization of a 20-bp oligomer, using washes in 6 x NaCl/citrate or 3.0 M Me4NCI.
AMV-RT = avian myeloblastosis virus/reverse transcriptase.
I
I
PCR = polymerase chain reaction.
PCR D E T E C T I O N OF D E N G U E VIRUS I N M O S Q U I T O
457
As seen in lane B of the 1.2 % agarose gel (fig. 1), PCR amplification of RNA from infected-mosquito midgut revealed a 289-bp band, the specificity of which was confirmed by Southern blotting and hybridization with a 20-bp oligomer which did not overlap those used for amplification (fig. 2), according to standard protocols (Wallace et al., 1981 ; Wood et aL, 1985). Thus, PCR amplification of material obtained from infected-mosquito midgut provided a straightforward and sensitive method of detection of dengue virus infection in a given tissue of a single mosquito. KEY-WORDS: Aedes albopictus, Dengue; PCR, Note.
RI~SUME UT!LISAT!ON DE L'AMPLIFICATIONGI~NIQUEPOUR LA DI~TECTIONRAPIDE DE PART1CULESVIRALESCHEZ LE MOUSTIQUE
Nous pr6sentons un protocole de PCR (amplification enzymatique g6nique), permettant la d~tection de particules virales de dengue au sein de cellules de l'6pith61ium intestinal du moustique vecteur, Aedes albopictus, apr6s infection par voie orale. MOTS-CLI~S: Aedes albopictus, Dengue; PCR; Note.
REFERENCES KUBERSKI, T.T. & ROSEN, L. (1977), A simple technique for the detection of dengue
antigen in mosquitoes by immunofluorescence. Amer. J. trop. Med. Hyg., 26, 533-537. SAIKi, R.K., GELFANO,D.H., STOFFEL,S., SCHARF, S.J., HIGUSHI, R., HORN, G.T., MULLIS, K.B. & ERLICH, H.A. (1988), Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491. WALLACE, R.B., JOHNSON, M.J., HIROSE, T., MIVAKE, T., KAWASHIMA, E.H. & ITAKURA,K. (1981), The use of synthetic oligonucleotides as hybridization probes. -- II. Hybridization of oligonucleotides of mixed sequence to rabbit B-globin DNA. Nucl. Acids Res., 9, 879-894. WOOD, W.I., GITSCHIER,J., LASKY,L.A. & LAWN, R.M. (1985), Base composkionindependent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc. nat. Acad. Sci. (Wash.), 82, 1585-1588.
