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The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO1, leprosy remains endemic in many countries with approximately 250,000 new cases each year.2 The entire M. leprae genome has been mapped3,4 and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites).5 Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci.5,6,7 Variable number tandem repeat (VNTR)5 analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing7,8,9, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA)10 has been used to study leprosy evolution and transmission in several countries including China11,12, Malawi8, the Philippines10,13, and Brazil14. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS).10 The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions.10 The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.
The purpose of this video article is to provide an overview of the work flow along with data format and interpretation for researchers that may just be starting this type of work (Figure 1). It includes demonstration of techniques, simplified protocols and practical tips described in previously published works.5,10
There should be at least 3 separate work areas for this kind of research. The laboratory should have 1) a pre-PCR area with a PCR hood (clean air box or isolated work area) for primer preparation (dilution, aliquot preparation and mixing), 2) a separate bio-safe cabinet for handling and addition of DNA to the PCR mixtures, and 3) a post-PCR work area for preparing and loading gels and for preparing samples for FLA. Primers and DNA samples should be kept in separate freezers and refrigerators. Primer contamination is one of the chief and most persistent problems in laboratory work of this type. Pipettes used for primers and PCR mixes should NOT be used for DNA. There should be separate sets of pipettes for the pre-PCR, PCR and post-PCR work areas. Generally, one researcher can process 12-18 samples in a 12-24 hour period using standard laboratory equipment.
Before beginning each phase of work:
Clinical samples containing M. leprae are obtained from leprosy patients who visit skin clinics. Routine diagnostic samples may be skin punch biopsies, slit skin smears or nasal swabs. Generally, punch biopsies or slits skin smears are the best for molecular epidemiology because they are clean and contain sufficient amounts of M. leprae. Use of these materials for research must be approved according to institutional guidelines.
When the combined primers are added to the PCR mixtures (Table 3), final concentrations drop to 0.2μM each.
*This protocol has been standard for many years and is an optional step that can be employed if confirmation of PCR products is desired prior to sending them for FLA.
Gel electrophoresis of the PCR products will hopefully produce a band for each locus in the primer combination (Figure 3). In Figure 3, there are 2 sections to the gel: the top section has Combination 1 PCR samples and the lower portion Combination 2 samples. Each section contains a 20 base pair molecular ladder, followed by PCR products obtained from 8 patient samples. Combination 1 also has a negative control and finally a positive control (NHDP63 strain). (The controls for combination 2 were on a different gel.) Note that most samples clearly display 5 bands, 1 for each locus in the combination. In some cases, bands may be too close together to appear as separate loci resulting in the appearance of only 4 bands.
Expected amplicon sizes are listed in Table 1. Our laboratory uses NHDP63 strain of M. leprae as a positive control. Two types of repeat segments have been studied: microsatellite loci (with 1-5 base repeats) and minisatellite loci (with segments greater than 5 base pairs repeated multiple times). 5
Interpretation of the data files from capillary electrophoresis relies on 2 standards: an internal DNA fragment sizing standard called GeneScan -500LIZ (ABI) and an external positive control sample of amplified bacterial DNA.
The FLA chromatograms, viewed using Peak Scanner software, can be seen in Figures 5a, 5b and 6.
Peak Scanner provides data on amplicon size (x-axis in base pairs) and signal strength (y-axis). (Figure 5b) Additional data on peak area, etc. are also available, although the size and peak height values are most important. Peaks less than 100 units in height are usually considered too weak a signal to be reliable.
Figure 6 compares the positive control (NHDP63) and two patient samples, showing a variation in the number of tandem repeats at locus (GTA)9. In the positive control, the sequence (GTA) is repeated 10 times. The NHDP63 VNTR and amplicon size were verified through gene sequencing. Patient 4's PCR amplicon is 3 bp smaller than the positive control indicating there are only 9 repeat units, while Patient 6 has an amplicon that is 3 bp larger than NHDP63 revealing 11 repeats of (GTA). Patient 2 had low bacterial index (BI) with little or no DNA PCR replication, therefore no FLA signal.
Difficulty in FLA data interpretation sometimes occurs as a result of 'stuttering'. During the PCR reaction, the DNA polymerase may produce fragments that are 1 or more repeats longer or shorter than the source allele. These are usually recognized as peaks of lower height surrounding the main peak. They will be 'on the ladder' that is, the correct number of base pairs of the repeat segment larger or smaller than the principal peak. The result is a family of peaks of the same color. Figure 7 shows this with locus (TA)10.
Another difficulty sometimes encountered with FLA involves '+A' or 'A-tailing', in which the DNA polymerase adds a single base [usually adenine (A)] to the 3' end of the copied DNA segment. This shows up in FLA as a peak to the right of a main or stutter peak that is 1 base pair larger than the adjacent peak as shown in Figure 8. It should not be confused with a main or stutter peak. A principal peak and its A-tail are considered a single species. The A-tail does not alter the number of VNTR repeats. (Some DNA polymerase kits are designed to specifically promote A-tailing in order to reduce this confounding effect. Promoting complete A-tailing tends to produce a single peak rather than a pair of peaks.)
The DNA extraction and PCR products contain both M. leprae and human DNA, however with the exception of (TA)18, the primers are specific enough that they only amplify the bacterial DNA, and there is little or no human DNA amplification. (TA)18 often produces a peak at 242 base pairs that is from human DNA and is not seen in armadillo passaged DNA samples (Figure 9).
The shelf life of primers is generally good provided they are kept at -20°C, only removed for immediate use, and then stored at 4°C until consumed. Primers should be stable at 4°C for 1-2 weeks. Even though making primer combinations for PCR is somewhat time consuming, it is recommended that only enough primer combination for immediate use be prepared. Primer combinations seem to degrade somewhat when stored for prolonged periods.
TE should be aliquoted from larger stocks, and aliquots may be stored either frozen or at room temperature. Larger aliquots of 200-400μl are good for diluting dried or concentrated primer stocks (Figure 2a). Smaller aliquots of 10-50 μl are useful for supplementing the volumes of primer combinations 3 and 4. TE is inexpensive and aliquots should be discarded after use.
Multiplex enzyme kits are quite expensive and should be kept frozen (-20°C) until use. Following PCR preparation, any unused multiplex solutions should be returned immediately to 4°C. The small Qiagen Multiplex PCR Kit comes with 3 tubes of multiplex mix, each containing 0.85ml (850μl) of solution; enough for about 65-70 PCRs.
Generally, it is best to avoid repeated, major temperature changes for the materials used in this type of laboratory work including the DNA samples, primers and multiplex kit solutions. All materials should be stored at -20°C until needed, and then kept at 4°C until consumed. Long term storage of DNA should be at -80°C.
All materials containing spent tissue, primers and/or DNA should be autoclaved and disposed of when no longer of any use. All samples treated with ethidium bromide or formamide should be treated as hazardous waste and disposed in accordance with the institution's hazardous materials policy.
Table 1: Amplicon sizes for strain NHDP63
Table 2: Cycling Parameters for VNTR PCR
Table 3: Preparation of PCR
Table 4: Allele Calls for Combination 1
Table 5: Allele Calls for Combination 2
Table 6: Allele Calls for Combination 3
Table 7: Allele Calls for Combination 4
Figure 1: VNTR-FLA Process Flow Diagram
Figure 2. (a) Preparation of Combination 1 Upper Primers (Eppendorf tube picture courtesy of www.clker.com). (b) PCR Setup (for 8 PCRs) (Eppendorf tube picture courtesy of www.clker.com)
Figure 3. Agarose gel of Combinations 1and 2 VNTR PCR product DNA
Figure 4. (a) FLA Plate Map. (b) FLA data files: *.fsa
Figure 5. (a) FLA Chromatogram of Positive Control (NHDP63) for Combination 1 VNTR loci. (b)Peak Scanner data for amplicon size and abundance of (GTA)9
Figure 6.Comparison of PCR samples to the PC (NHDP63) for locus (GTA)9
Figure 7.Main and Stutter Peaks for (TA)10
Figure 8: +A (A-tail) Peaks Adjacent to Main or Stutter Peaks.
Figure 9: (TA)18 Main Peak, Stutter Peaks and Human DNA Peak
Figure 10: (A) Strain Differentiation of M. leprae based on VNTR data. (B) Strain Differentiation of M. leprae based on MLVA DNA Fingerprint
The collection of skin samples from leprosy patients requires skilled clinicians or technicians working at skin clinics. Laboratory workers handling these samples must take great care to wear lab coats, gloves and protective eye wear and to work in a bio-safety cabinet when handling infected samples of human or armadillo tissue. Disinfection of surfaces and tools is also critical. Working in a clean, sterile bio-safe cabinet is important for avoiding contamination of DNA samples.
DNA extraction has become relatively easy thanks to the development of extraction kits from companies like Qiagen. Directions must be followed carefully. All samples should be kept cold when not in use. Avoid repeated, extreme temperature changes for samples.
DNA primers used in this work can be ordered from various companies which have been cited in the list of literature references at the end of this paper. Care should be taken to work with primers in a DNA free environment in order to avoid contamination. Primers come as dry powder and must be mixed with TE and diluted to a 100μM concentration, then separated into smaller quantities (Figure 2a). Working solutions of primers are further diluted to 10μM concentrations. Again, do not use DNA samples in areas/hoods where primers are used and prepared.
PCR products should also be kept cold when not in use.
A 3% agarose gel preparation will give better DNA band separation, but takes longer to run. For purely qualitative purposes, 2% is usually sufficient. Ethidium bromide solution used for staining the DNA in agarose gels is a highly active genotoxin that is absorbed through the skin. Handle this solution and gels stained with it using great care and always being sure to wear gloves. Wash hands thoroughly after handling any DNA samples or ethidium bromide materials. Dispose of ethidium bromide staining solution, wash and gels according to institutional guidelines. Gels are not routinely required; this step can be eliminated once FLA methods have been established. It is time and reagent consuming.
The formamide solution used in preparing samples for FLA is also highly toxic and should be handled with care. Wash hands after its use. Dispose of it following institutional guidelines.
Reading the results of FLA using Peak Scanner software can be challenging. A basic set of allele calls (number of tandem repeats) has been developed at CSU (Tables 4-7). (It should be noted that Tables 4-7 may not be all inclusive. As other strains of M. leprae are studied, alleles with copy numbers outside the ranges listed may be found.) One particular challenge involves 'stutter' peaks. These are families of peaks, especially of STRs that only involve 2-3 base pair repeats, such as (TA)10. Sometimes selecting the correct peak to read is difficult (Figure 7). In this figure, the peak in the positive control at 190 bp is the main peak. Peaks lower in height that are 2, 4, or even 6 base pairs larger or smaller than the main peak are called 'stutter peaks.' A-tailing can also cause confusion. A-tailing is described earlier in the text and in Figure 8. Finally, if PCR products are highly abundant in the samples, peaks may appear with a double spike. In this case, read the center of the peak form. (See Figure 6, Patient 6.)
Data management can be a formidable task for this type of work. It is important to record all pertinent information for all experiments such as: date of a task or procedure, operator, strip/plate maps, FLA orders, PCR conditions and recipes, dates of gels and photographs, storage temperatures, DNA template dilutions, FLA electronic file storage locations, etc. Good organization and data management can save hours of time spent looking for specific pieces of information in the future.
The laboratory work described here has been going on for several years at Colorado State University and at other locations around the world. The large picture of what all of the collected data means and how it may be of future use is beginning to emerge. Figures 10A and 10B demonstrate how these DNA fingerprints can be used to distinguish different M. leprae strains between countries (10A) or even between families (10B). Family and community linked cases have been shown to carry M. leprae of similar or identical VNTR strain types. The hope is that it may be possible to gain further insight into the mode(s) of leprosy transmission so that an early detection system of transmission networks may be developed for those people most at risk, and that curative drug therapy may begin before permanent neurological and dermatological damage is done.
No conflicts of interest declared.
Funding was provided by NIH/NIAID grant RO1-AI-63457 and ARRA grant supplement RO1-AI-63457 S1. We acknowledge the contributions of all current and past members of the laboratory group and collaborators.