[page 37↓]

2. Material and Methods

The polymerase chain reaction (PCR) was selected as the most appropriate method, with the best potential to achieve direct, sensitive and species specific diagnosis. In order to establish the methods, samples had to be collected first. It appeared that dermal scrapings from CL lesions, preserved on filter paper, would be a suitable sampling procedure for this purpose (Harris et al ., 1998; Färnert et al ., 1999). Apart from dermal scrapings several other specimens were obtained too, as for example Giemsa stained smears, paraffin embedded biopsies, peripheral blood, aspirates, spleen and liver tissue and also cultures of promastigotes. Samples were collected from patients as well as from various animal species, which were either known or suspected reservoir species of leishmaniasis. The collected and processed samples are presented here as groups . Detailed description of the samples is given together with the results in Tables 5-10.

2.1. Origin of samples:

2.1.1. Reference strains:

Preliminary studies were performed on purified DNA from various strains of all available species. These strains had been collected over many years and were preserved at the WHO-reference center for Leishmania strains at the Kuvin Center, Jerusalem. The cultured strains had been characterized previously by EF, isoenzyme analysis and PPIP-PCR (Eisenberger and Jaffe, 1999). The purified DNA of these strains was used for positive controls in every experiment. The New World strains were kindly provided by Linda Oskam, Royal Tropical Institute, Amsterdam. Table 2 shows the reference strains used in this study.

Table 2 : Reference strains

Species

LRC

WHO code

Source

Origin

L.major*

LRC-137

MHOM/IL/67/Jericho II

human

Jericho

L.major

LRC-465

IPAP/IL/84/UVDA

sandfly

Israel (Uvda)

L.major

LRC-509

MHOM/IL/86/BLUM

human

Jordan Valley

L.tropica*

LRC-36

MHOM/IQ/66/L75

human

Iraq

L.tropica

LRC-682

MHOM/TR/95/Urfa40

human

Turkey (Urfa)

L.tropica

LRC-747

IL/98/LRC-L747

sandfly

Israel (Kfar Adumim)

L.donovani*

LRC-133

MHOM/ET/67/HU3

human

Ethiopia

L.donovani

LRC-661

MHOM/SD/??/Khartoum

human

Sudan

L donovani

LRC-751

MHOM/IN/93/BI2302

human

India

L.d.infantum*

 

MHOM/TN/80/IPT1

human

Tunisia

L.d.infantum

LRC-716

MCAN/IL/97/LRC-L716

dog

Israel (Nataf)

L.d.infantum

LRC-719

MCAN/IL/97/LRC-L719

dog

Israel (Rishon le Zion)

L.aethiopica*

LRC-149

MHOM/ET/72/L102

human

Ethiopia

L aethiopica

LRC-495

MHOM/ET/85/Vasa

human

Ethiopia

L.gerbilli*

LRC-652

MRHO/CN/60/LD3

Rhombomys opimus

China

L turanica

LRC-654

MRHO/MN/83/MNR2

Rhombomys opimus

Mongolia

L arabica

 

MPSA/SA/83/JISH220

Psammomys

Saudi Arabia

L killicki*

 

MHOM/TN/80/LEM 163

human

Tunisia

L.mexicana

 

MHOM/GT/86/GO22

Oskam/Evans

 

L.braziliensis

 

MHOM/CO/81/MORO

Oskam/Evans

 

L.amazonensis*

WR 669

MHOM/BZ/73/M2269

no information

 

L.guyanensis*

WR 677

MHOM/BR/75/M4147

Oskam/WR

 

L.panamensis

 

MHOM/CR/87/NEL3

Oskam/lab

 

L.chagasi

 

MHOM/BR/74/PP75

LF Schnur

 

T.cruzi

 

-

LF Schnur

 

T.lewisi

T22

-

LF Schnur

 

L.seymourii

L524

-

LF Schnur

 

C.fasciculata

L466

-

LF Schnur

 

P.davidii

ATCC 50166

-

LF Schnur

 

All strains were part of the WHO Reference Center for Leishmania strains at the Kuvin Center. Parts of the collection had been cultivated at the Kuvin Center (coded with LRC- Leishmania reference center, animal or human source known). Other strains had been received from others, as for example the internationally used WHO-reference strains (*) (often no information on the source of collection). All strains were identified by isoenzyme electrophoresis, many of them also with additional methods (PCR, EF).

2.1.2. Patients:

Samples were collected from patients visiting the Dermatology clinic of the Hadassah Hospital with lesions suspected of CL. Over a period of more than one year 100 skin scrapings from Hadassah-patients were collected (by Flory Jonas). Skin scrapings and/or cultures were collected from 11 Patients of the Tel Hashomer Hospital, 6 of them had contracted the disease in the New World. Most of the examined patients were Israelis who either live in endemic areas, who had been travelling inside or outside the country or who had been stationed as soldiers in the Judean Desert or in the Negev.

A new focus of CL was discovered in early spring 2000 in a village named Wadi Albethan (West Bank/Samaria) , located between Nablus and the Jordan Valley. In 8 households skin scrapings and cultures were collected. Skin scrapings were taken from 23 patients on filter paper and on slides. Eleven lesion aspirates were collected for culturing in NNN and semi-solid media. The number of cultures was smaller since needle aspirations were not performed on facial lesions of infants, and some other individuals refused. Detailed information on single patients is presented in in chapter 3 (Results).

2.1.3. Animal samples:

Desert rodents:

An extensive field study on desert rodents was carried out by Gideon Wasserberg (PhD student of the Department of Life Sciences, Ben-Gurion-University of the Negev). Rodents were trapped in endemic areas of the Negev (Qziot and Nizana), and skin scrapings were collected from the ears for microscopical examination. With the beginning of the cooperation the tissue was collected on sterile filter paper as well (for PCR).


[page 39↓]

The predominant species was the fat sand rat ( Psammomys obesus ), besides gerbils ( Meriones crassus and Gerbil dasyarus ) were trapped and examined. This collection of animal tissue samples provided an excellent study group for testing the reliability of the PCR from tissue preserved on filter paper. DNA extraction and PCR was performed on ear scrapings of 28 individuals of P.obesus , 6 M.crassus and 19 G.dasyarus .

Canids:

Blood of four infected dogs was examined for the presence of leishmanial DNA. A drop of collected peripheral EDTA-blood was preserved on filter paper (received from Dr. Gad Baneth, School of Veterinary Medicine of the Hebrew University, Rehovot). The dogs had been diagnosed previously by microscopy on Giemsa stained lymph node and spleen aspirates.

In an earlier study by the same collegue jackals had been trapped in several areas in central Israel. The jackals had been previously screened for the presence of antibodies against L.d.infantum by ELISA and western blotting, partly with borderline positivity. No method for the detection of the antigen had been applied yet. Since PCR was available now it seemed to be worth-while to extract and amplify DNA from tissue of these animals in order to complete the survey. Ears and spleens were examined. The ears of 20 jackals were deep frozen at a temperature of –70°C. The spleens of 14 individuals had been preserved in buffered formalin . This was not very promising for good PCR results but it was worth-while to examine.

Hyrax:

Since hyraxes were highly suspected serving as reservoir for L.tropica (Photo 19), it was of interest to examine hyrax tissue. A parched body of a hyrax was found next to a cave in which a sandfly infected with L.tropica (LRC-747) had been trapped a few days earlier. The cave was close to Kfar Adumim, an Israeli settlement in the Judean Desert, one of the emerging L.tropica foci in the country. Most human cases had occurred in the periphery of the settlement, facing the slope with the caves (Photo 13). The presence of fecal remains of hyraxes as well as the corps indicated that these caves were inhabited by hyraxes. Several specimens of the hyrax were extracted, namely bone powder from the femur, tissue from the skin and nose. Ear tissue was not available. One ear sample from a living hyrax, which had been trapped in the Galilee (by Gunther Müller, Kuvin Center), was examined too.

2.1.4. Cultivation of Leishmania promastigotes:

Cultures were obtained by Lionel Schnur (Kuvin Center, Hebrew University) and Flory Jonas (Dermatology Clinic, Hadassah Hospital), or collected during visits (Wadi Albethan, Tel [page 40↓] Hashomer Hospital). One culture derived from a sandfly trapped in a cave close to Kfar Adumim, one of the major L.tropica foci in the country (Alon Warburg , Kuvin Center ).

2.2. Laboratory work:

2.2.1. Sampling:

The study focused on dermal scrapings preserved on filter paper. Skin scrapings were obtained from patients with cutaneous lesions and from rodents. After desinfection with 70% ethanol a small incision was cut with a disposable surgical blade radially into the lesion, and some tissue was scraped off as described by Herwaldt, (1999). It was important to obtain tissue with as little blood as possible. The material was blotted on UV-radiated and autoclaved 3 mm filter paper (Schleicher and Schüll, Germany) and on glass slides for Giemsa staining. The tissue samples were wrapped with aluminium foil and stored at room temperature. Prior to extraction, the scraped tissue spots were cut off of the filter paper using sterile surgical blades. A new blade was used for each sample. The tissue samples were transferred to screw-cap 1.5 ml tubes containing either 500 m l NET buffer, 500 m l guanidine thiocyanate solution or 250 m l chelex (5%), depending on the extraction method used.

2.2.2. Giemsa staining of smears:

Smears were collected on glass slides, air dried and fixed with methanol for a few seconds. Giemsa stain was filtered and diluted 1:20 with phosphate buffer (pH 7.2). After 20 minutes of staining the slides were washed with tap water and air dried. The stained smears were examined under the microscope with a 40x lens and with a 100x oil immersion lens. If at least one intra- or extra-cellular amastigote with a distinctive kinetoplast was found the smear was declared positive. When no amastigotes were seen after 15 minutes the smear was declared negative. Microscopy of the smears of the Hadassah patients and of the desert rodents was performed by Flory Jonas and Gideon Wasserberg respectively. Many of the patient smears and some of the rodent smears were double checked, the observations were in concordance. All samples collected during visits (Wadi Albethan, Tel Hashomer Hospital) were stained and examined in our laboratory.

2.2.3. Collection and cultivation of Leishmania strains:

Cultures were obtained by needle aspiration from the border of the lesions. Usually it was necessary to inject about 0.1 ml of sterile 0.9% saline into the lesion in order to aspirate a drop of fluid as described by Herwaldt, (1999). The aspirated fluid was discharged into two culture tubes, one containing semisolid (SS) blood agar (Schnur and Jacobsen, 1987) and the other NNN [page 41↓] medium (Novy-MacNeal-Nicolle medium) overlayed with Schneider's (Gibco, Grand Island, NY; Biological Industries Beit Haemek, Israel) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 200 µg/ml streptomycin, and 200 U/ml penicillin. The cultures were incubated at 24-28°C. After a few days one drop of media was examined under the microscope. If promastigotes were found the cultures were transferred into Schneider’s Drosophila medium for further culturing. The parasites were cultivated in 5 ml of Schneider’s medium for 2-3 days before they were transferred to a larger volume (40 ml). Negative cultures were examined again several times and in case of no growth they were discarded only after 3 weeks. DNA-extraction was performed from 40 ml of densely grown parasites (see later).

2.2.4. DNA extraction:

Several different methods were tested, in order to find reliable and simple methods which could be adopted in a clinical laboratory and also be employed for epidemiological studies (high number of samples).

Phenol-chloroform-extraction:

1. Extraction from clinical samples, mostly dermal scrapings preserved on filter paper:

Each tissue sample was transferred into a 1.5 ml tube and lysed with 500 m l of NET buffer (50 mM NaCl; 10 mM EDTA; 50 mM Tris-HCl). Sodium dodecyl sulfat (SDS) was added to a final concentration of 5 %. RNAse was added to a final concentration of 100 m g/ml (5 m l of a 10 mg/ml solution) and the samples were incubated at 37 ° C for 30 minutes. Proteinase K (10 m l of a 10 mg/ml solution) was added to a final concentration of 200 m g/ml. The samples were digested over night, rotating in a hybridization oven at 60 ° C. Equal volumes of saturated phenol were added to each sample (500 m l). The samples were mixed gently for 5 minutes. After centrifugation at high speed (12000 rpm) for 5 minutes at room temperature the upper phase was carefully removed and transferred to new 1.5 ml tubes. A phenol and chloroform mix (chloroform/isoamylalcohol 25:1) was added in equal volumes to each sample (250+250 m l). The samples were mixed gently and centrifuged for 3 minutes. The upper phase was again transferred to a new tube. Equal volumes of chloroform were added and the samples were centrifuged for 1 minute. The upper phase was once again transferred. The DNA samples were precipitated with 50 m l of 3M sodium actetate (NaAc 300 mM, pH 6.0) and one volume (500 m l) of cold (-20 ° C) isopropanol and then stored on ice for 30 minutes. Subsequently, the samples were centrifuged at 14,000 rpm for 45 minutes at 4 ° C. The isopropanol was removed and the DNA pellets were washed with cold (-20 ° C) 70% ethanol (250 m l). The samples were centrifuged for 10 minutes. [page 42↓] The ethanol was removed, and the DNA pellets were vacuum dried. The DNA samples were then resuspended in 25 m l of sterile water and dissolved for one hour at 56 ° C. The fresh DNA samples were immediately subjected to PCR experiments. The remainder of the DNA was aliquoted and stored at -20 ° C.

DNA extraction from cultures:

The procedure routinely performed at the Kuvin Center has been described by Eisenberger and Jaffe, (1999). The reference strains used as positive controls had been extracted by this method as well as every newly collected strain which was cultivated during this research. Parasites were harvested at a density of about 2 x 10 7 parasites/ ml. The cells were centrifuged for 10 minutes at 2500 rpm and washed three times with phosphate bufered saline (PBS). Proteinase K digestion and phenol-chloroform extraction followed. The protocol differed in some points from the extraction described above: Triton-X 100 (1%) was used instead of SDS. The proteinase K digestion was performed for 2h. The DNA was resuspended in 100 m l of TE buffer (1 mM EDTA; 10 mM Tris-HCl; pH 8.0). The DNA concentrations were determined photometrically at an absorbtion of 260 nm and stored at 4°C. Dilutions of 100 ng/µl and 20 ng/µl were prepared.

Guanidine extraction:

The method has been described by Boom et al. , (1990).

Preparation of silica beads:

Six g of silica beads (Sigma) were washed in 50 ml of sterile ddH 2 O and mixed gently for 24 h at room temperature in a rotating hybridization oven. After a quick centrifugation the water was pipetted off. The silica beads were washed once again, the second time rotating for 5 h was sufficient. Sixty m l of HCl (1M) were added to the pellet. The consistency of the silica beads was viscous. The purified silica beads were transferred to a brown glass tube, autoclaved and stored at room temperature. The prepared silica beads were stable for long term use.

Extraction:

The tissue samples were transferred to 1.5 ml tubes containing 500 µl of guanidinium thiocyanate solution (4M guanidinium thiocyanate (GTC), 0.1 M Tris-HCl pH 6.4; 0.02 M EDTA, pH 8; 1.3% Triton X-100) and incubated over night at 56 ° C under gentle agitation. One ml of 6 M sodium iodate (NaI) and 10 µl of resuspended silica beads were added. The samples were vortexed and kept on ice for one hour. After a quick centrifugation (3 seconds at 12,000 rpm), the supernatant was carefully removed and discarded. Wash buffer (0.01M Tris-HCl, pH 7.5; 0.05 M NaCl; 1 mM EDTA pH 8; Ethanol 50%) was added to each tube. The silica bead [page 43↓] pellets were vortexed until they were in suspension. Another quick centrifugation followed and the buffer was removed. The pellets were washed with 100% ethanol and vacuum dried. They were resuspended in 100 µl of sterile ddH 2 O and incubated for one hour at 56 ° C. The silica beads were spun down prior to PCR to avoid any inhibitory activity. The guanidine solution and the sodium iodate were freshly prepared for every new extraction. The wash buffer was stored at -20 ° C.

Chelex extraction:

A 5% Chelex solution was prepared by washing 500 mg of Chelex -100 (Biorad) in 10 ml of sterile ddH 2 O. The suspension was boiled for 10 minutes in a water bath. After centrifugation for 5 minutes at 3000 rpm the water was removed. The washing step was repeated once. The purified chelex was resuspended in 10 ml of ddH 2 O and aliquoted to 250 m l volumes for subsequent use. The samples were incubated over night at 56° and then heated for 10 minutes to 94 °C. After centrifugation of 1 minute at 12000 rpm the supernatant was directly used for PCR (Harris et al., 1998).

Crude methods:

Lysis of cultured parasites:

PCR diagnosis from cultured strains was performed whenever new strains were cultivated in the laboratory and a differentiation between L.major and L.tropica was required. The density of parasites was not defined, since cultures were usually subjected to PCR as soon as there was a microscopical evidence of promastigotes. Cultures (100 µl) were transferred to 1.5 ml tubes and diluted 1:4 (300 µl) with ddH 2 O. The cultures were boiled for 5 minutes to increase cell lysis. One µl of the lysate was used as template.

Crude preparation of dermal scrapings on filter paper:

Dermal scrapings were processed as for the phenol-chloroform extraction. After the over night digestion with proteinase K the samples were only heated for 10 minutes to 95°C (or boiled for 5 minutes) to inactivate the enzyme and then centrifuged shortly at high speed (12,000 rpm). The supernatant was directly used for PCR.

2.2.4.1. Choice of extraction methods according to specimens:

The patient samples (dermal scrapings on filter paper) were tested with each of the presented extraction methods. Some of the other sample groups were extracted with one specific method: The phenol-chloroform method was used for the jackal samples and the hyrax ear. A small part [page 44↓] (~0.5 g) of each jackal ear was chop p ed with a surgical blade and transferred to a 1.5 ml tube. Small pieces (~0.1 g) of the formalin fixed jackal spleens were cut out and were washed three times with sterile PBS prior to the extraction. Thorough purification was also required for Giemsa stained smears and paraffin embedded skin biopsies (phenol-chloroform). The guanidine extraction method was used for several series of patients samples (dermal scrapings on filter paper) and also for the parched hyrax tissues. The chelex extraction method was employed on dermal scrapings from patients and on the rodent samples. The crude extraction methods were used experimentally on filter paper samples from patients.

2.2.4.2. Extraction from paraffin embedded biopsies:

Four paraffin embedded skin biopsies of CL patients were proceeded. For deparaffinization, several slices were cut from each block and transferred into a 1.5 ml tube, containing 1 ml of xylene. The sample was vortexed and then left for 10 minutes at room temperature. After centrifugation for 5 minutes at 12,000 rpm the xylene was removed and the process repeated. One ml of absolute ethanol was added, the samples were mixed and centrifuged for 5 minutes and the ethanol was removed. The ethanol washing was repeated twice. Finally, the samples were vacuum dried, redisolved in ddH 2 O and submitted to phenol-chloroform extraction (Laskay et al ., 1995).

The 3 extraction methods (phenol-chloroform, guanidine and Chelex) were compared on infected mouse liver tissue, which had been evenly distributed on filter paper. Squares of 0.5 cm 2 were cut off the filter paper (6 pieces for each method, always 2 stored at a different temperature, see also the following passage).

2.2.4.3. Storage conditions:

The positive extraction control (mouse liver infected with L.donovani ) was distributed homogenously on 3 pieces of filter paper. Each filter paper was stored at a different temperature (-20 ° C, 4 ° C and 20 ° C), in order to test the stability of the DNA in the tissue over a period of 1 year under different conditions. Squares of the same size (0.5 cm square) were cut from each filter paper and extracted in doublets by phenol-chloroform-, guanidine and chelex extraction.

2.2.5. PCR:

2.2.5.1. Oligonucleotides:

In the literature no primers were found which could achieve sensitive and species specific diagnosis for all species simultaneously. In order to adapt a diagnostic system to the specific [page 45↓] needs of the region, 3 different pairs of oligonucleotides were employed, which targeted at the kinetoplast minicircles (Uni21/Lmj4; 13A/13B and MP3H/ MP1L). The kinetoplast minicircles were selected as a suitable target due to the high copy number (10.000), implying the potential for high sensitivity. Close to the end of the study another pair of primers ( LITSRn/L5.8S) was published (El Tai et al., 2000) and introduced also to this work. These primers target the intergenic transcribed spacer (ITS) and fulfill all the requirements (sensitive and species-specific diagnosis).

Whole minicircle amplification with primers Uni21/Lmj4:

The primer pair (Uni21/Lmj4) had been specifically designed for the purpose of direct diagnosis and the differentiation of L.major and L.tropica in Israel, which was had been the initial aim of this study. This primer pair had not been tested previously and was suggested by Prof. Greenblatt, Kuvin Center. The primer pair Uni 21 (5' -GGGGTT GGTGTAAAATAGGCC - 3') and Lmj4 (5' - CTAGTTTCCCGCCTCCGAG - 3') was designed by Eresh, Axelrod, Greenblatt and Barker (Eresh et al., 1993). The primer design was based on a published sequence from a L.major minicircle (Smith et. al ., 1989). Primer Uni 21 was selected from a sequence within the conserved region. Primer Lmj4 was designed on the basis of the variable region of the same minicircle sequence.

2. Genus specific kinetoplast primers 13A/13B:

For the screening of desert rodents another PCR was employed, which needed to be primarily sensitive but not necessarily species specific. For this purpose kDNA primers 13A/13B (Rodgers et al ., 1990) appeared to be suitable. Primers 13A (5'-GTGGGGGAGGGGCGTTCT-3') and 13B (5'-ATTTTCCACCAACCCC CAGTT-3') amplified a sequence of 120 bp of the kDNA minicircles. This sequence is conserved in all minicircle classes of all Leishmania species. Due to the small size of the sequence and its presence in all minicircles the PCR with primers 13A/13B is highly sensitive and therefore suitable for screening.

The following Figures demonstrate how primers Uni21/Lmj4 and primers 13A/13B relate to the minicircle, as a sequence in Figure 6 and as a circle in Figure 7.


[page 46↓]

Figure 6: Section of the L.major minicircle

Figure 7: kDNA minicircle with primers Uni21/Lmj4 and 13A/13B

L.braziliensis specific kinetoplast primers MP3H/ MP1L:


[page 47↓]

Primers MP3H (5’-GAACGGGGTTTCTGTATGC-3') and MP1L (5’- TACTCCCCGACATG

CCTCTG-3') were developed by Lopez et al ., (1993) in order to distinguish between the species of the L.braziliensis and the L.mexicana complex. A 70 bp sequence specific of L.braziliensis minicircles is amplified. The L.mexicana complex as well as all Old World Leishmania species are not amplified.

Amplification of the ITS-1 region with primers LITSRn/L5.8S:

This non-kinetoplast based PCR has been recently published by El Tai et al ., (2000). Primers LITSRn (5'- CTGGATCATTTTCCGATG - 3') and L5.8S (5'- AAGTGCGATAAGTGGTA-3') amplify a section of the intergenic transcribed region (ITS-1, about 300 bp) of all Leishmania species. In combination with the enzyme digestion of the PCR product using the restriction enzyme Hae III (= BsuRI ) species specific diagnosis is achieved. ITS sequences of L.d.infantum, L.major, L.tropica, L.braziliensis, L.guyanensis, L.panamensis, L.mexicana, L.amazonensis and of other Leishmania species are published in the EMBL data base. These sequences are species specific and restriction sites could be identified that would allow the differentiation of all important Leishmania species, in the case of L.donovani and L.braziliensis the identification of the complex. (G. Schönian, personal communication). Figure 8 shows the ITS region and how primers LITSRn/L5.8S relate to it.

Restriction of the ITS-1 PCR-product:


[page 48↓]

The ITS-1 PCR products were digested with BsuRI (HaeIII) (MBI Fermentas, Lithuania). The amplified PCR products (8 m l) were mixed with 1 m l of enzyme and 1 m l of 10x buffer (10 mM Tris-HCl, pH 8.5; 10 mM MgCl 2 ; 100 mM KCl; 0.1 mg/ml BSA) in 0.5 ml tubes, and were incubated for 2 hours at 37 ° C .

2.2.5.2. PCR-conditions:

The regular volume of each PCR reaction was 25 m l. Only the ITS-1 amplifications were either performed in 25 or in 50 m l. The reaction mixtures were prepared on ice. All components were first pipetted into a 1.5 ml tube, well mixed and then aliquoted to 200 m l PCR tubes. For positive controls, 1 m l (20 ng/ m l) of purified leishmanial DNA was used. When resuspended DNA from filter paper extractions was examined, 1 to 5 m l of extracted DNA were added. In case of lysed cultures 1 m l was sufficient as template. The reaction mix without DNA served as negative control.

Several different polymerases were employed. The Promega Taq polymerase (Promega, Madison, WI) was used as a standard enzyme for most of the reactions. It was generally employed for the PCR with primers 13A/13B and MP3H/MP1L and for initial studies with primers Uni21/Lmj4 on purified DNA. For the studies on dermal scrapings with primers Uni21/Lmj4 a polymerase with a higher activity and stability was employed in order to increase the chances for positive results. In the beginning the TaKaRa Ex Taq polymerase (Takara Shuzo co., LTD., Japan) was used. Later the Fermentas Taq polymerase (MBI Fermentas, Lithuania) proved to be more reliable and was adopted in the laboratory.

The reaction mix using the Promega Taq polymerase was composed of 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1 % Triton X-100, 1.5 mM MgCl 2 , 200 m M of each nucleotide (dATP, dGTP, dCTP, dTTP), 1 m M of each primer, 1 U of polymerase and up to 5 m l of template DNA. Primers MP3H/ MP1L required only 0.625 U of the polymerase, since the amplified sequence was smaller (70 bp). PCR with the TaKaRa Ex Taq polymerase was performed in 10x Ex Taq buffer, 2 mM MgCl 2 , 200 m M of each nucleotide, 1 m M of each primer, 0.5 U of TaKaRa Ex Taq polymerase and up to 5 m l of template DNA. The PCR with the Fermentas enzyme was performed in 75 mM Tris-HCl (pH 8.8), 20 mM (NH 4 ) 2 SO 4 , 0.01% Tween 20, 1.5 mM MgCl 2 , 200 m M of each nucleotide, 1 m M of each primer, 1 U of Fermentas Taq polymerase and 1-5 m l of template DNA. Table 3 gives details on the 4 different PCR methods used in this study.


[page 49↓]

Table 3: PCR methods

1. PCR with primers Uni21/Lmj4:

 

   

reagent

original conc.

m l

final conc.

 

ddH 2 O

 

15.0-19.0

 

 

buffer

10 x

2.5

1 x

 

Mg 2+

25 mM

1.5

1.5 mM

 

Primer Uni21

100 m Mol

0.25

25 pmol/25 m l

 

PrimerLmj4

100 m Mol

0.25

25 pmol/25 m l l

 

dNTP’s

20 mM

0.25

0.2 mM

 

Taq-Polymerase

5 U/ m l

0.25

0.625 U/ 25 m l

 

DNA

 

1-5

 

 

final volume

 

25

 

 

2. PCR with primers 13A/13B:

   

reagent

original conc.

m l

final conc.

ddH 2 O

 

13.5-17.5

 

 

buffer

10 x

2.5

1 x

 

Mg 2+

25 mM

1.5

1.5 mM

 

Primer 13A

100 m Mol

1.0

100 pmol/25 m l

 

Primer 13B

100 m Mol

1.0

100 pmol/25 m l

 

dNTP’s

20 mM

0.25

0.2 mM

 

Taq-Polymerase

5 U/ m l

0.2

1U/ 25 m l

 

DNA

 

1-5

 

 

final volume

 

25

 

 

3. PCR with primers MP3H/MP1L:

   

reagent

original conc.

m l

final conc.

 

ddH 2 O

 

13.5-17.5

 

 

buffer

10 x

2.5

1 x

 

Mg 2+

25 mM

1.5

1.5 mM

 

Primer MP3H

100 m Mol

1.0

100pmol/25 m l

 

PrimerMP1L

100 m Mol

1.0

100pmol/25 m l pmol/25 m l

 

dNTP’s

20 mM

0.25

0.2 mM

 

Taq-Polymerase

5 U/ m l

0.125

0.625 U/ m l

 

DNA

 

1-5

 

 

final volume

 

25

 

 

4. PCR ITS with primers LITSRn/L5.8S

   

reagent

original conc.

m l

final conc.

 

ddH 2 O

 

33.0-37.0

 

 

buffer (Promega)

10 x

2.5

1 x

 

Mg 2+

25 mM

3

1.5.mM

 

Primer LITSRn

100 m Mol

1.0

25 pmol/50 m l

 

PrimerL5.8S

100 m Mol

1.0

25 pmol/50 m l

 

dNTP’s

20 mM

4.0

0.2 mM

 

Taq-Polymerase

5 U/ m l

0.4

2U/50 m l

 

DNA

 

1-5

 

 

final volume

 

50

 

 

Cycling was performed in a minicycler (M.J. Research, Watertown, USA). Mineral oil was not required because the machine was equipped with a heating device from above, in order to prevent evaporation within the PCR tubes. Table 4 shows the different cycling protocols for each PCR-method (as finally established).

2.2.6 Agarose gels:

1.5% or 2.5% agarose gels were prepared by dissolving 1.5 g respectively 2.5 g of agarose (Seakem LE agarose, FMC Bioproducts, Rockland, Maine, USA) in 100 ml of TAE buffer (0.04 M Trisacetate; 0.0001 M EDTA) in an Erlenmeyer flask . The percentage of agarose depended on the size of the PCR product. For the amplificates with primers Uni21/Lmj4 (680-850 bp) a 1.5% agarose gel and for the amplificates with primers 13A/13B (120 bp) and MP3H/MP1L (70 bp) a 2.5% gel was prepared . The agarose was dissolved by boiling in a microwave oven for 2-4 minutes, until the solution was transparent. After cooling the agarose to about 60-70 ° C ethidium bromide (2.5 m l) was added. The well mixed fluid was poured into a gel-cast (DNA plus, USA Scientific) and was left to solidify.

For higher resolution, 5 m l of Gel star stain (FMC BioProducts, Rockland, Maine) was used instead of ethidium bromid. Gel star stain was employed for the analysis of the restriction patterns obtained after digestion of the ITS-1 PCR-products (3% agarose gel) as well as for the amplification of filter paper samples with primers Uni21/Lmj4 (the least sensitive primers).


[page 51↓]

Table 4: Cycling

 

1. PCR with primers Uni21/Lmj4:

Preheating

5 min

94 ° C

 

Denaturation

1 min

94 ° C

35-45 cycles

Annealing

80 sec

60 ° C

Elongation

1 min

72 ° C

Extension

10 min

72 ° C

 



2. PCR with primers 13A/13B:

Preheating

3 min

94 °

 

Denaturation

1 min

94 ° C

 

30 cycles

Annealing

1 min

50 ° C

Elongation

1 min

72 ° C

Extension

10 min

72 ° C

 



3. PCR with primers MP3H/MP1L:

Preheating

3 min

94 ° C

 

Denaturation

1 min

94 ° C

 

35 cycles

Annealing

1 min

54 ° C

Elongation

1 min

72 ° C

Extension

10 min

72 ° C

 



4. PCR of the ITS-1 region with primers LITSRn and L5.8S:

Preheating

4 min

95 ° C

 

Denaturation

40 sec

95 ° C

 

36 cycles

Annealing

30 sec

53 ° C

Elongation

1 min

72 ° C

Extension

6 min

72 ° C

 


[page 52↓]

Ten μm l of each PCR product were loaded with 2.5 m l of 5x bromophenol blue loading buffer (BPB) on the gel. A few m l (2-3 m l) of molecular size markers PGEM, f HAE or f Hinf (100 ng/ μm l) were loaded as controls. PGEM was used for the larger PCR-products, f HAE or f Hinf were used for the small amplificates. Electrophoretic separation was performed at 120 V and 120 mA for about 40 minutes. The PCR products were visualized under UV-light and photographed (ImageMaster VDS, Biotech Pharmacia). The pictures were captured with the NIH-Image program.

2.2.7. Avoiding inhibition:

Several experiments were performed using additives in the PCR, in order to reduce possible inhibition caused by substances, which were not sufficiently eliminated by the crude extraction (such as hemoglobin). Bovine serum albumin (BSA) or dimethyl-sulfoxyd (DMSO) in combination with formamide were used. BSA was sterile filtrated and used in a concentration of 10 mg/ml. The effect of BSA was examined by adding 0, 1, 2 and 4 m μl to 4 reactions performed with 1 m l of crude template.

A similar experiment was performed using 2.5 % DMSO and 1% formamid in three reactions, containing 1, 2.5 and 5 m μl of crude template. As a comparison, 3 reactions without additives, with 1, 2.5 and 5 m μl of crude template were prepared and amplified.

2.2.8. Avoiding contamination:

The potential sensitivity of the PCR was theoretically up to less than one parasite. This implied a high risk for contamination, which had to be strictly avoided. As a general rule, the extraction of DNA, the preparation of the PCR and electrophoresis of the PCR products should be strictly separated. It is also recommended to use different sets of pipettes for the different parts of the work. Due to limitations of space and technical equipment this standard could not be met. Instead, extra precautions had to be taken:

-DNA extraction from clinical material and preparation of the PCR was performed in a hood, located in a Leishmania -free room. The parts of the work, which were less sensitive and which were by themselves a potential source of contamination, were performed in a separate room (PCR-cycling, gel electrophoresis and also the extraction from cultured parasites)

-Gloves were changed during the procedures from time to time.

-The working surface in the hood was bleached with 5% bleach and UV-radiated prior to extraction or PCR.


[page 53↓]

-Before every new extraction or PCR the pipettes were wiped with 5% bleach and UV-radiated from both sides for at least 15 min. This was necessary, because only one set of pipettes was available, which was used alternately for highly sensitive purposes and for very concentrated DNA (PCR- amplified or extracted DNA from cultures).

-The micro-centrifuge was bleached prior to extractions since the tops of the 1.5 ml tubes came in contact with the upper margin of the cups holding the tubes. Since normally highly concentrated DNA was centrifuged in the same centrifuge these cups were assumed to be a source of contamination.

-A security distance between the 1.5 ml tubes was kept by placing the tubes in distances to each other. By this, the tops of neighbouring tubes did not touch each other when opened.

-Filter tips were used for extraction and PCR.

-Whenever possible 20 m l tips were used instead of 10 m l tips. Due to the length of the 20 m l tips the shaft of the pipette could not touch the 1.5 ml tubes from inside. For volumes below 4 m l the 10 m l pipette was used to ensure accuracy.

-For extraction tight tubes had to be used since they were to rotate in a hybridization oven. Only screw-cap 1.5 ml tubes proved to be tight enough for this purpose.

2.2.9. Controls:

1. Negative controls:

The extraction as well as the PCR had to be monitored strictly for possible contamination. Two negative extraction controls were routinely used for every extraction series. A piece of sterile filter paper and a piece of filter paper with a drop of human blood (without history of previous infection with leishmaniasis) were extracted parallel to the samples. In order to exclude non-specific results in the rodent samples, ear tissue from a negative Psammomys was used as negative control. The tissue was collected from a laboratory animal of the Diabetes Unit of the Hadassah Hospital, Ein Karem (received from Dr. Ehud Ziv). For the other animal species no suitable negative controls were available. The PCR itself was monitored by one reaction in every PCR, which contained only the reagents and no template.

2. Positive controls:

Liver tissue of a laboratory mouse, which had been infected with L.donovani , was blotted on filter paper. A piece of about 0.5 cm in diameter was used as a positive control for the extraction. As an amplification control and also as a reference, 20 ng of DNA from at least one of the Leishmania reference strains was amplified in every experiment.


[page 54↓]

2.2.10. Solutions:


[page 55↓]


[page 56↓]

2.2.11. List of reagents:

Agarose (LE agarose)

BsuRI (HaeIII)

Chelex-100 Resin

dNTP

Gelstar nucleic acid stain

Giemsa's staining solution

Guanidine-thiocyanate

L-cystine dihydrochloride

L-tyrosine disodium

Molecular size markers (PGEM, f HAE, f Hinf)

Oligonucleotides

Proteinase K

RNase A

Schneider's Drosophila medium powder

Silica beads

Sodium iodate

Taq polymerase

Taq polymerase

Taq polymerase (TaKaRa Ex Taq)

Triton X-100

Yeastolate (bacto-yeast-extract)

 

Seakem, Rockland Maine, USA

MBI Fermentas, Lithuania

Bio-Rad

MBI Fermentas, Lithuania

FMC BioProducts, Rockland, Maine

Gurr, BDH, England

Fluka

Sigma

Sigma

Promega

Genset SA, France

Sigma

Sigma

Gibco

Sigma

Merck

MBI Fermentas, Lithuania, Germany

Promega, Madison, Wi

TaKaRa Biomedicals, Shuzo CO., LTD, Japan

BDA

Difco

 


© Die inhaltliche Zusammenstellung und Aufmachung dieser Publikation sowie die elektronische Verarbeitung sind urheberrechtlich geschützt. Jede Verwertung, die nicht ausdrücklich vom Urheberrechtsgesetz zugelassen ist, bedarf der vorherigen Zustimmung. Das gilt insbesondere für die Vervielfältigung, die Bearbeitung und Einspeicherung und Verarbeitung in elektronische Systeme.
DiML DTD Version 3.0Zertifizierter Dokumentenserver
der Humboldt-Universität zu Berlin
HTML generated:
08.12.2003