Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease

Molecular and Biochemical Parasitology, 33 (1989) 205-214 Elsevier

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MBP 01107

Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle D N A sequences: use in diagnosis of Chagas' disease Nancy R. Sturm 1, Wim Degrave 2, Carlos Morel 2 and Larry Simpson 1 1Department of Biology and Molecular Biology Institute, University of California, Los Angeles, CA, U.S.A. 2Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil (Received 12 September 1988; accepted 21 October 1988)

Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids, kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects. Key words: Trypanosoma cruzi; Minirepeat; Polymerase chain reaction; Minicircle DNA; Schizodeme

Introduction C h a g a s ' d i s e a s e affects 12 m i l l i o n p e o p l e in the A m e r i c a s , with a p p r o x i m a t e l y 32 million p e o p l e at risk of i n f e c t i o n , a n d t h e r e is no g e n e r a l l y useful c h e m o t h e r a p e u t i c t r e a t m e n t o r vaccine. T h e d i s e a s e is c a u s e d by t h e k i n e t o p l a s t i d p r o t o z o a n , Trypanosorna cruzi, which is t r a n s m i t t e d cyclically by insects of the R e d u v i i d family or via b l o o d t r a n s f u s i o n f r o m an i n f e c t e d d o n o r . In the m a m m a l i a n host, a f t e r an initial a c u t e p h a s e , the p a r a s i t e m i a m a y l a p s e into a l o n g - l a s t i n g c h r o n i c p h a s e , with e n s u i n g m u l t i s y m p t o m a t i c a n d p o l y m o r p h i c s y n d r o m e s . D u e to t h e low a b u n d a n c e

Correspondence address: L. Simpson, Department of Biology and Molecular Biology Institute, University of California, Los Angeles, CA 90024, U.S.A. Abbreviations: kDNA, kinetoplast DNA; PCR, polymerase chain reaction; SDS, sodium dodecylsulfate.

of circulating t r y p o m a s t i g o t e s in the b l o o d , the m o s t r e l i a b l e m e t h o d o f direct p a r a s i t o l o g i c a l det e c t i o n of this infection in the c h r o n i c state is xen o d i a g n o s i s [1], which entails allowing u n i n f e c t e d l a b o r a t o r y t r i a t o m i d s to t a k e b l o o d m e a l s f r o m the p a t i e n t , and r e q u i r e s an i n c u b a t i o n t i m e of s e v e r a l weeks. O t h e r m e t h o d s i n c l u d e axenic culture of b l o o d a n d infection of s u s c e p t i b l e l a b o r a t o r y animals. I d e n t i f i c a t i o n a n d classification o f the p a r a s i t e into z y m o d e m e s [2,3] o r schizod e m e s [4] r e q u i r e s analysis of i s o e n z y m e p a t t e r n s and k i n e t o p l a s t minicircle D N A r e s t r i c t i o n enz y m e d i g e s t i o n profiles. B o t h m e t h o d s r e q u i r e m o r e t h a n l 0 s cells, which a r e o b t a i n e d by o u t g r o w t h of the p a r a s i t e s . H o w e v e r , the o u t g r o w t h of p a r a s i t e s in insects, c u l t u r e o r a n i m a l s m a y incur selection of p a r t i c u l a r strains p r e s e n t in the original p o p u l a t i o n , thus raising the possibility that the results o b t a i n e d d o n o t a c c u r a t e l y reflect the original p o p u l a t i o n of p a r a s i t e s in t h e p a t i e n t [5,6].

0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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T. cruzi cells, like other kinetoplastids, contain a complex network of catenated circular D N A molecules (termed kinetoplast D N A or kDNA) within their single mitochondrion [7]. Each network consists of 5-20 x 103 minicircles of 1.42 kb and 20-50 maxicircles of 36 kb. The minicircles consist of multiple sequence classes, the exact number and frequency of which is uncertain. Each minicircle is organized into four 120 bp minirepeat conserved regions situated at 90 ° intervals and four non-repetitive variable regions characteristic of that sequence class [8]. In spite of the high copy number of k D N A minicircles per cell, the use of labeled oligonucleotide probes complementary to conserved regions of the minicircles to detect and classify T. cruzi cells into schizodemes proved impractical with low numbers of parasites (unpublished results). In this paper we show that the polymerase chain reaction (PCR) [9] can be used to amplify species- and strain-specific fragments of k D N A minicircles for detection and classification of small numbers of T. cruzi cells in the presence of a vast excess of human DNA. Materials and Methods

DNA isolation. A cloned minicircle insert from the C1 strain of T. cruzi (pTc-21) [8] was used as a homogeneous, linear template for template titration and animal blood lysate reconstructions. k D N A was prepared by a modification of the methods of Simpson [10] and Saucier et al. [11]. Cells were washed in 0.1 M E D T A , 0.15 M NaC1, 10 mM Tris-HCl (pH 7.9) and resuspended at 1.2 × 10 9 cells m1-1. Sarkosyl was added to 3% and pronase to 0.5 mg m1-1 and the lysate incubated at 60°C for 1-3 h. The iysate was then syringed through a #18 needle at 25 psi and centrifuged for 2 h at 24000 rpm in the SW 28 rotor. The crude kDNA pellet was dissolved in 10 mM Tris-HCl, 1 mM E D T A (pH 7.9) and layered on a step gradient of cesium chloride, prepared by layering 6 ml of cesium chloride with 100 ~xg ml 1 ethidium bromide ( n D = 1.4040) under 24 ml of cesium chloride without dye ( n D = 1.3705) in an SW 28 polyallomer tube. The tube was centrifuged for 15 min at 20 000 rpm and the k D N A at the lower interface visualized by UV illumination and recovered by tube puncture. The dye was removed

by n-butanol extraction, and the k D N A precipitated with ethanol. For some experiments involving the use of extremely low template D N A concentrations, purified networks were briefly sonicated in a Braunsonic 1510 sonicator to liberate minicircles. The k D N A from several other kinetoplastid genera was purified by the same method: Crithidia luciliae, Leptomonas collo-

soma, Herpetomonas mariadeanei, Endotrypanum sp., Blastocrithidia culicis, and Leishmania tarentolae. Whole cell D N A from cultured Trypanosoma rangeli was isolated by standard procedures [12].

PCR amplification of minicircle sequences. The k D N A samples (intact or sonicated) were used as templates for PCR using Thermus aquaticus (Taq) D N A polymerase [13] in an amplification buffer composed of 10 mM Tris-HC1 (pH 8.8), 50 mM KC1, 1 mM dithiothreitoi, and 1.5 mM of each deoxytrinucleotide. The 50 txl reactions were manually cycled through water baths in 400 ixl Eppendorf tubes as follows: The first cycle consists of a 2 min denaturation step at 94°C, 2 min annealing at 37°C, addition of 1-4 U of Taq DNA polymerase, and 1-2 min elongation at 70°C; subsequent cycles consist of 1 rain at 93°C, 1 min at 37°C, and 1-2 min at 70°C. Elongation times were 1 rain for conserved region amplification and 2 min for variable region amplifications. Samples were covered with 75 Ixl light mineral oil. Prior to sampling, the denaturation step was omitted; reactions were cooled at 37°C for 1 min before drawing the sample. Samples (1/10 of the reaction volumes) were run on either 4% NuSieve agarose gels, 10% acrylamide gels, or 2.2% low melting agarose gels.

Oligonucleotides. Oligonucleotides were synthesized by standard phosphoramidite methods. The sequences and locations of the oligonucleotides used as primers and probes are shown in Fig. 1. The Cll and Y1 oligonucleotides cover polymorphic regions of the Cll minirepeat (region 4) and the Y01 minirepeat (region 1) [8]. Hybridization of blots. Reaction products were blotted and fixed onto Nytran (Schleicher and Schuell) filters by cross-linking with short wave-

207

A l0

20

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Consensus: A A A a t t G G G g N t N N G A A A T T c N G G A A A N T I q T G G T T T T G G G A G G G G C G T T d A A a T T T t G G d g C S67: 5'-TGGTTTTGGGAGGGGC~ T T C A A ~ T T T 70

80

90

100

1]O

120

GgAAATTCATGCATCTCCCCCGTACATTATTTtGgCNAAAATGgGGATTTTTcaNGG~AGGT S 33 A: 5'-T C A T G C A T C T C ~ C C C G T A C A T T A T T T S35 :A G T A C G T A G A G ~ G G G C A T G T A A T A A A 5' C I1:5'-T G C G G A T T T T G G G A T T T T T - A C G G G G A G Y I:5'-G G T C G A A A A T G G G G - T T G T T T A C 130

140

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.

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GGGGTTCGATTGGGGTTGGTGTAATATAGNNANtNNNNTGg S 34 A: C C A A G C T A A C C C C A A C C A C A T T A T A T 5' $36: 5'-G G G T T C G A T T G G G G T T G G T G T

B

83 bp S33A

;Z2bp T( ~ S34A s67 {

/s36 °o,'

ee e

33Obp Fig. 1. (A) The consensus nucleotide sequence of 20 minirepeat regions as determined by Degrave et al. (fig. 4 in ref. 8) for five sequenced minicircles from three strains of T. cruzi. Uppercase letters indicate a level of matches of more than 60%, lowercase letters indicate a level between 50 and 60%, and N (any nucleotide) indicates levels of less than 50%. The locations and sequences of the oligonucleotide primers used are indicated in their proper orientation. Degeneracies were chosen based on nucleotide polymorphisms in the aligned minirepeats [8]. (B) A schematic representation of the organization of the minirepeat regions on an individual minicircle with the primer sets and their amplification products indicated by dotted lines. The size of the minicircle is approximately 1.42 kb. Note that only a single primer set and product is indicated for each minirepeat for simplification. l e n g t h u l t r a v i o l e t light for 2 min. H y b r i d i z a t i o n a n d wash c o n d i t i o n s u s e d for 32p-labeled oligon u c l e o t i d e p r o b e s a r e as d e s c r i b e d in W o o d et al. [14], m o d i f i e d b y o m i t t i n g d e x t r a n sulfate f r o m t h e h y b r i d i z a t i o n s o l u t i o n a n d using 1% s o d i u m d o d e c y l s u l f a t e ( S D S ) in t h e wash solution. T h e filters w e r e r o u t i n e l y w a s h e d at 45°C a n d t h e n at 65°C in 3.0 M t e t r a m e t h y l a m m o n i u m c h l o r i d e solution [14] with 1% S D S ; this m e t h o d e l i m i n a t e s b a s e r a t i o effects a n d allows o l i g o m e r h y b r i d i z a tions to b e p e r f o r m e d at i d e n t i c a l stringencies.

Isolation of DNA from blood. T o t a l D N A f r o m h u m a n b l o o d , a n d f r o m T. cruzi i n f e c t e d m o u s e b l o o d was r e c o v e r e d by the high s a l t - d e t e r g e n t lysate m e t h o d of Z o l g et al. [15], f o l l o w e d by phenol/chloroform deproteinization and ethanol p r e c i p i t a t i o n . 400 p,1 of a m i x t u r e of w a t e r / l % N a s a r k o s i n a t e in 50 m M E D T A ( p H 8.0)/cesium trif l u o r o a c e t a t e (2.04 g m1-1, P h a r m a c i a F i n e C h e m i c a l s , Inc.) (2:1:1, v/v) was a d d e d to 100 ~1 o f b l o o d . T h e s e l y s e d b l o o d s a m p l e s w e r e stable at r o o m t e m p e r a t u r e . T h e lysate was e x t r a c t e d with an e q u a l v o l u m e o f p h e n o l / c h l o r o f o r m (1:1)

208 and the D N A was precipitated with ethanol from the aqueous phase. In the case of the infected mouse blood samples, half of the total D N A recovered from each 100 Ixl sample was used as template for the P C R analysis. Results

Selection o f oligonucleotide primers for PCR. Degrave et al. (fig. 4 in ref. 8) aligned 20 minirepeats from five minicircles which were cloned from the CL, Y and A W P strains of T. cruzi. On the basis of these alignments, and with the assumption that the observed conserved sequences within the minirepeats are present in most if not all minicircles in the k D N A networks, three conserved regions were selected for synthesis of oligonucleotide primers, as shown in Fig. 1A. A schematic diagram of the three expected amplification products on a minicircle containing these conserved sequences is shown in Fig. lB. Note that the 83 and 122 bp products contain overlapping minirepeat sequences, whereas the 330 bp

A 1

product covers the adjacent variable region sequences in addition to a portion of the minirepeat. Since each variable region abuts a minirepeat, the 330 bp product should contain all variable region sequences from all minicircles in the network. The expected 83, 122 and 330 bp products were obtained by amplification of T. cruzi k D N A with the appropriate primer sets, as shown in Fig. 2A and B. In addition, an approximately 50 bp ethidium bromide-stained band of variable intensity is present in the no-template D N A primer control lane 1 and also in lanes 2, 4 and 6. This band represents a primer hairpin artifact, the intensity of which is inversely proportional to the amount of template D N A used in the reaction (see Fig. 3 below). The 720 bp band in Fig. 2B is most likely also an artifact produced by hairpinning of the 330 bp product, since this band hybridizes to the $67 probe (Fig. 2B) but not to the Cll probe (data not shown), as would be expected if this product represented the amplification of two adjacent variable regions.

B 2

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M

CI Peru Y

CI Peru Y

330 bp 122 bp 83 bp

Fig. 2. (A) Amplification of the 83 and 122 bp fragments from three strains of T. cruzi. Lane 1, no template DNA, $34A/$67 primer control; lanes 2 and 3, CL strain kDNA template; lanes 4 and 5, Peru strain kDNA; lanes 6 and 7, Y strain kDNA; lane M, 123 bp ladder DNA marker. The amount of template DNA in each reaction was 15 pg. The products were electrophoresed in a 4% NuSieve agarose gel which was stained with ethidium bromide. (B) Amplification of the 330 bp fragments from three strains of T. cruzi. The products were electrophoresed in a 2.2% low melting agarose gel which was blotted and probed with 32p_ labeled $67 oligonucleotide.

209 #rninicircles: I0,000 1000 lO0 50

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work containing 104 catenated minicircles is straightforward.

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Fig. 3. Template titration using purified insert DNA from minicircle clone pTc-21 for the S33A/S34A amplification. A 2.2% low melting agarose gel of the PCR products was blotted and probed with 32p-labeled C11 oligonucleotide. The approximate number of template molecules calculated from the DNA concentration is given above each lane. The lane marked 0 represents a no template DNA primer control. Lane M, 123 bp DNA ladder.

Sensitivity of PCR amplification of minicircle fragments. In order to ascertain the sensitivity of the PCR amplification reaction with non-catenated minicircle template DNA, cloned pTc-21 minicircle insert DNA from the C1 strain was used in a template titration experiment with primers $33A and $34A, as shown in Fig. 3. The 83 bp product was visualized both by ethidium staining and by hybridization with the internal strain-specific oligonucleotide probe, Cll. The presence of at least two approximately 50 bp ethidium bromide-stained artifact bands can be seen in all lanes, including the no template DNA control. The minimal amount of template DNA required to visualize the 83 bp product after 50 cycles of PCR was 0.075 fg by ethidium bromide staining, and 0.015 fg by hybridization with the C11 internal probe; this is the equivalent of approximately 0.1% of the minicircle content of a single T. cruzi cell [16]. It is clear that detection of a single net-

Amplification is T. cruzi species-specific. DNA from the closely related species, T. rangeli [17], yielded a product of equivalent size when amplified with the 122 bp primer set and this product hybridized with the internal conserved probe, $33A (Fig. 4A), but not with the strain-specific internal probes, Cll and Y1 (data not shown). However, no product was produced when T. rangeli kDNA was amplified with the variable region primer set, $35/$36 (Fig. 4B). kDNA networks (10 ng) from several other kinetoplastid protozoa - Blastocrithidia culicis, Endotrypanum sp., Leishmania tarentolae and Leptomonas collosoma - showed no amplification products after 30 cycles PCR with primer sets S33A/S34A or $34A/$67 (data not shown). C. luciliae and H. mariadeanei kDNA networks, however, yielded discrete amplification products with primers $34A/$67 and S33A/S34A, but these products were of much lower relative intensity and different sizes than the products from T. cruzi kDNA (Fig. 4A), and did not hybridize with the $33A probe. Amplification is T. cruzi strain-independent. kDNA networks from three strains of T. cruzi, the C1, Peru and Y strains, each yield 83 or 122 bp fragments on amplification with the S33A/S34A or the $67/$34A primer set, respectively (Fig. 2A). The 330 bp variable region amplification product, which hybridizes with the $67 probe, is also obtained from all three strains using the $35/$36 primer set (Fig. 2B). Strains of T. cruzi can be differentiated by hybridization of amplified fragments with specific probes or by digestion of the amplified variable region with restriction enzymes. Oligonucleotide probes Cll and Y1 specific for polymorphic regions internal to the 122 bp region (Fig. 1) were used to distinguish strains by hybridization to blots of the amplified products (Fig. 5A). The strains could also be distinguished as different schizodemes by restriction enzyme digestion patterns in 10% acrylamide gels of the amplified 330 bp variable region fragments (Fig. 5B). The patterns observed

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¸ il ~!~ .....

Fig. 4. (A) Con-familial controls for amplification of the 122 bp minicircle fragment. Total cell DNA (200 ng) from T. rangeli (lane 1) and 10 ng kDNA from C. luciliae (lane 2), H. mariadeanei (lane 3), and T. cruzi (lane 4) were used as templates with the primer set $34A/$67. The gel was blotted and probed with 32P-labeled $33A oligonucleotide. Lane M, 123 bp DNA ladder. (B) Lack of amplification of the 330 bp variable region fragments with T. rangeli DNA. Total cell DNA from T. rangeli (1.1 p,g) (lane 1) and T. cruzi Peru strain (60 pg) (lane 2) and kDNA from the T. cruzi CI strain (30 pg) (lane 3) were used as templates for amplification of the 330 bp variable region fragments.

were reproducible with several preparations, implying that the bands represent complete digestion products. Presence o f excess h u m a n D N A has no effect on amplification. A 3.6 billion fold mass excess of

human D N A did not interfere with amplification of the 122 bp product, using T. cruzi C1 strain k D N A as template, as shown in Fig. 6. The presence of additional lower molecular weight ethidium-stained bands present in the primer alone lanes is probably due to self-priming of the oligonucleotides. These bands are more intense when the initial template D N A quantity is low. Additional higher molecular weight bands also occur in the h u m a n D N A control lanes. The T. cruzi-specific 122 bp amplified fragments could be identified by hybridization of the blot with the internal Cll p r o b e (Fig. 6). Reconstruction experiments using rabbit blood and cloned pTc-21 minicircle insert D N A were performed to determine the sensitivity of detection of non-catenated minicircle D N A by P C R after re-isolation from blood. The minicircle D N A

was added to Zolg blood lysates, and total D N A was then isolated and used as template. As few as approximately 50 template molecules in a 100 ~1 sample of blood sufficed for subsequent gel detection of the amplified 122 bp fragment by hybridization of the blot with the Cll probe (data not shown). Detection o f T. cruzi cells in infected m o u s e blood.

Samples of blood (100 ill) from mice experimentally infected with the Peru strain of T. cruzi were analyzed for the presence of parasites by isolation of total D N A and amplification of the 122 bp T. cruzi minicircle fragment (Fig. 7), Positive amplification results were obtained with as few as ten trypanosomes in a 100 Ixl sample, as calculated from the h e m o c y t o m e t e r counts. A positive result was obtained with a blood sample which showed no trypanosomes in 30 h e m o c y t o m e t e r fields. This mouse eventually died of the infection, suggesting that the P C R method is m o r e sensitive than direct counting of parasites. Another mouse, which also showed no trypanosomes in 30 hemocytometer fields at this time and

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B Enzyme. No Enzyme TuqI RseI MboI Strain. M ~CI Peru Y~ rCI Peru Yq rcl Peru y1 rCI Peru y1

492 bp369bp-

A Strain: CI Peru Y

246 bp-

Etd 123bpCll

Y1 Fig. 5. Distinction of three strains using PCR amplified minicircle fragments. (A) Amplified 122 bp fragments from the C1, Peru and Y strains separated in a 4% NuSieve agarose gel, which was blotted and probed with 32p-labeled strain-specificoligonucleotides, CI1 and Y1. (B) Digestion of the 330 bp amplification products from three strains of T. cruzi with several enzymes, as resolved on a 10% acrylamide gel. M, 123 bp DNA ladder. which later died from the infection, was negative for amplification of the 122 bp fragment (Fig. 7). We interpret the latter negative result as due to the extremely low circulating parasitemia at this time of infection; larger blood samples would be required to detect circulating parasites at this level of infection. T h e r e is no technical reason, however, that the isolation procedure could not be scaled up. Since the precise level of circulating parasites in blood of chronic Chagasic patients is unknown, the a m o u n t of blood required to detect

parasitemia by the P C R method in such patients remains to be determined. Discussion P C R amplification of specific k D N A minicircle fragments provides a sensitive and specific method for detection and classification of T. cruzi cells. A large excess of host D N A does not interfere with the reaction and does not give rise to T. cruzi-specific amplification products, and the

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Kinetoplast HumanDNA and Human Kinetoplast Primer Control DNA DNA Control M r30 35 401r30 35 401r30 35 401r30 35 40 ~

122 bp

-122 bp 4q

Fig. 6. Amplification of the 122 bp minicircle D N A fragment in the presence of a vast excess of h u m a n D N A . The D N A template was 6 fg sonicated T. cruzi network D N A mixed with 21.4 p,g purified h u m a n D N A . The 4% NuSieve agarose gel was blotted and probed with 32p-labeled Cll oligonucleotide. The lanes marked 'Primer Control' and ' H u m a n D N A Control' represent samples which contain primer D N A but no template k D N A .

kDNA template can be easily and efficiently recovered from blood lysates using host nucleic acids as carrier. We have targeted for amplification two overlapping fragments within the con-

served region of the minicircle minirepeat and one fragment that covers the adjacent variable region and a portion of the minirepeat. The amplification of these fragments is specific for T. cruzi, ex-

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t-~22bp

Fig. 7. Infected mouse blood samples analyzed for the presence of T. cruzi parasite DNA by PCR amplification of the 122 bp minicircle fragments. (A) Reaction products using the $34A/$67 primer set after 30 cyclesof PCR for blood samples from different infected mice in which parasites were seen in hemocytometer. (B) Reaction products after 50 cyclesof PCR for blood samples from two infected mice in which no parasites were seen in 30 fields in the hemocytometer; both mice eventually died from T. cruzi infection. The products were separated in a 2.2% low melting agarose gel, which was blotted and probed with 32p-labeledCll oligonucleotide.The lanes marked 0 in A and B represent uninfected mouse blood DNA controls. M, 123 bp marker DNA ladder.

product can be digested with restriction enzymes to provide a qualitative schizodeme profile comparable to those obtained by digestion of total k D N A [4] and could also be probed with strainspecific variable region probes. A more complete knowledge of the extent of minicircle sequence variation within a single strain as well as a knowledge of minicircle sequence variation between strains would provide important information for the construction of strain-specific minicircle oligomer libraries for analysis of amplification products, and this work is in progress. Currently employed assays for T. cruzi such as xenodiagnosis [1], zymodeme analysis [2,3] and schizodeme analysis of total k D N A [4] require some form of culture of the parasite, either in insects, cell cultures or animals, which may cause selection of particular strains of the parasite from a multiply infected host. The PCR method avoids this problem by direct analysis of small numbers of parasites from the host without outgrowth. Oligonucleotide primers are used which have been shown to produce PCR products from a variety of T. cruzi strains (Fig. 2, and Avila, Sturm, Degrave, Goncalves and Simpson, unpublished resuits). The high salt lysate blood sampling method of Zolg et al. [15] proved useful for obtaining samples of infected blood for later analysis. This amplification method should also prove useful for analysis of small numbers of trypanosomes in tissues of mammalian hosts and in infected insects.

Acknowledgements cept for the case of the closely related species, T. rangeli [18], which also shows amplification of the conserved region fragments. The T. rangeli kDNA can, however, be distinguished by using the variable region primer set, since this k D N A was found to lack template activity for variable region amplification, the reason for which is unknown. The absence of strain-specificity in the amplification of the three minicircle fragments implies that these products can be used as diagnostic markers for T. cruzi. Additional information as to the specific strain can be obtained by use of internal strain-specific probes covering polymorphic regions, such as the Cll and Y1 probes, or, better, by analysis of the amplified 330 bp variable region product. The variable region

We thank Dr. Christian Orrego for originally suggesting the use of PCR for detection of T. cruzi, Dr. Jerry Manning for supplying the infected mouse blood samples, Dr. W. Zolg for communication of unpublished results, Dr. George Holz for supplying kinetoplastid cell cultures, Dr. James Dvorak for supplying T. rangeli cells, Dr. A. Goncalves for supplying k D N A samples, Dr. A.M. Simpson for communication of unpublished results, and Janet Shaw for assistance and discussions. This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases to L.S. and C.M.

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