7-6 Identification by DNA probes and primers

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What metabolic tests are really sensing is the absence or presence of a set of enzymes inside the microbe that carry out a specific function. All enzymes are encoded in the genome (the DNA) of the microbe. It is therefore possible to detect the presence of the genes that codes for specific enzymes instead of detecting the metabolic products of that enzyme activity. DNA detection methods depend upon the hybridization of a short piece or pieces of synthesized DNA to the genome of the test species. These short pieces of DNA are called probes or primers depending upon the method. The DNA genome of the test species is extracted, often by simply boiling a small bit of culture of the microbe, and then exposed to the primer. Once bound, the primer is detected in some fashion, the most common method being by PCR. DNA detection has several advantages.

  • Depending upon how the primer is designed and what the target is, the test can be specific to a species or even a subspecies, or it can detect multiple microbes. For example, a primer can be made to the shigga-like toxin of certain strains of E, coli, specifically identifying heamorrhaggic E. coli. On the other end of the spectrum, there are primers that have been created to conserved regions of the 16S rRNA of bacteria that will detect any bacteria present in a sample.
  • A mutation that would inactivate an enzyme, and fail in a biochemical test, will often still be positive in an DNA detection method.
  • With a well designed probe or primer a single DNA test can identify a microbe. Identification by metabolic tests requires a large number of tests to narrow it down to the species level.

Until a few years ago, detecting the presence of specific DNA sequences was much more difficult than performing metabolic tests. Even though there are advantages to using DNA tests, they remained something done in research labs for very different purposes. The emergence of PCR and specifically Real Time PCR (RT-PCR) is beginning to break down that barrier. Using RT-PCR, it is possible to detect the presence of a microbe in a sample in minutes to hours. Microbes can be detected at very low levels, sometimes just a few cells per gram of sample are detectable.

Real time PCR is a modification of the PCR protocol such that PCR fragments are rendered detectable by fluorescence, thus it become unnecessary to run an agarose gel to determine the absence or presence of a DNA fragment. Many kits are available to detect pathogens in food. However, UW-Madison uses a modified protocol that simulates the results one would obtain from a kit. Why are we not using a kit? Two reasons. First, they are expensive, costing $300-400 for 100 samples. Second, they have very high specificity. For example, the kit for hemorrhagic E. coli detects the labile toxin, which is responsible for many of the symptoms of the disease. Therefore, only the bona fide pathogen will work in the assay. While this is a good thing in the field, in a teaching laboratory it would require that you be given the real pathogen; probably not a good idea. Especially with hemorrhagic E. coli as it is believed that the infectious dose can be as low as 10 microorganisms.

In our assay, fluorescence is generated by the binding of SYBR green, a dye developed by Invitrogen. SYBR green is excited by light at 498 nm and emits light at 522 nm when returning to the ground state. The dye is highly specific for double stranded DNA, and has a much higher fluorescence when bound to it.

In RT-PCR, a PCR reaction is run in the presence of SYBR green. If a positive template is present, the PCR primers bind to it and create double stranded DNA (dsDNA). The SYBR green dye binds to this dsDNA. As the amplification proceeds, the concentration of dsDNA:SYBR green complex increases with the concentration of dsDNA. Specialized machines, RT-PCR thermocyclers, have been developed to measure the fluoresce coming out of PCR reactions after each cycle. If a positive template is present, a strong SYBR green fluorescence will be observed in the tube. Conversely, samples without a positive template will have a lower fluorescence. One caveat, SYBR green is non-specific and will bind to any dsDNA fragment present in solution. It is therefore important to perform a melting curve after the amplification to verify that the fragment obtained is the correct sequence. DNA sequences will have a unique temperature at which they melt, with the melting temperature dependent upon the specific primary sequence of the fragment. In most RT-PCR reactions, the correct fragment will have a unique melting temperature compared to any false positives that may have arisen in the tube. With a carefully designed experiment and given the right primers, it is possible to test for the presence or absence of any DNA sequence and therefore any desired microbe or set of microbes. Below is given a general protocol for extracting DNA and testing for various bacteria using RT-PCR. This protocol was adapted from the references listed at the end of this page.

Procedure

For a food or other natural sample, perform the following.

  1. The sample must first be washed and centrifuged at low speed to remove large particulates before harvesting the microbes. Resuspend 0.1 g of sample in 3 ml Buffer 1 (50 mM sodium phosphate buffer [pH 8], 0.1% Tween 80). Vortex vigorously for about 30 seconds.
  2. Spin the sample at 200 x g for 15 minutes. Carefully pipette off the supernatant into an Oakridge centrifuge tube and place the tube on ice
  3. Repeat steps 1 and 2 two more times on the original sample, pooling approximately 9 ml of supernatant.
  4. Centrifuge the 9 ml of supernatant at 15,000 x g @ 4°C for 10 minutes to pellet the cells. Resuspend the cells in 100 µl of sterile water.

For a colony to be tested, perform the following

  1. With a sterile loop, pick a single colony and resuspend it in 100 µl of sterile water.

For a broth or enrichment culture

  1. Remove 100 µl of sample and place it in a microcentrifuge tube. Centrifuge at 14,000 rpm (or at high enough velocity to pellet the cells) for 20 seconds. Resuspend the cell pellet in 100 µl sterile water.

Once processed continue from here

  1. Boil the sample at 100 °C for 10 minutes.
  2. Chill the sample on ice for 10 minutes. Centrifuge in a table top centrifuge for 1 minute and collect the supernatant. This liquid will serve as your source of template DNA.
  3. Prepare your real time PCR tube according to the following table.

    Master mix is purchased from Promega corporation. It comes as a 2x concentration. The SYBR green dye is purchased from invitrogen. (the material for gel staining) and comes in a 10,000x concentration. It is used in PCR assays at 100,000 x dilution. A 10x solution is a 10,000-fold dilution of the SYBR green. The primers are used as a concentration of primer is 0.25 µm. We obtained the primers from genosys.

    PCR test for Tm (°C) primer forward (µl) primer reverse (µl) master mix template DNA (µl) SYBR green dye (10x) (µl) double distilled water Melt temp
    E. coli60 1.7 1.6 25 10 5 to 50 µl 87.5
    Salmonella 55 2.4 2.9 25 10 5 to 50 µl 86
    Listeria 2 1.8 25 10 5 to 50 µl 80

    Below are the sequences of the primers used to detect each species, along with their expected length of the fragment that should be generated if amplification is successful.

    E. coli Primers (340 bp)
    F: 5'-GTTAA TACCT TTGCT CATTG A-3'
    R: 5'-ACCAG GGTAT CTAAT CCTGT T-3'
    Salmonella primers (85 bp)
    F: 5'-CCTTT CTCCA TCGTC CTGAA-3'
    R: 5'-TGGTG TTATC TGCCT GACC-3'
    Listeria monocytogenes primers (98 bp)
    F: 5'- TCCGC AAAAG ATGAA GTTC-3'
    R: 5'-ACTCC TGGTG TTTCT CGATT-3'
  4. The following thermal cycling conditions are an initial DNA denaturation step at 95 °C for 10 min followed by 45 cycles of denaturation at 95 °C for 15 s, primer annealing at the optimal temperature for 40 s, extension at 72 °C for 60 s.
  5. Finally, melt-curve analysis was performed by slowly heating the PCRs to 95 °C (0.3 °C per cycle) with simultaneous measurement of the SYBR Green I signal intensity.

References

  1. Jothikumar N, Wang X, Griffiths MW Real-time multiplex SYBR green I-based PCR assay for simultaneous detection of Salmonella serovars and Listeria monocytogenes. J Food Prot. 2003 66(11):2141-5.
  2. Erja Malinen, Anna Kassinen, Teemu Rinttila and Airi Palva (2003) Comparison of real-time PCR with SYBR Green I or 59-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology (2003), 149, 269-277

Figure 7-15 Running RT-PCR experiment

Samples are processed as described in the protocol and then mixed with template DNA, taq polymerase, dNTPs and primers. Each complete reaction is pipetted into in a 96-well rack (a) and placed into a RT-PCR system. The University of Wisconsin-Madison Bacteriology Department uses the Applied Biosystems 7500 Real-Time PCR System (b). The system is programmed using their software. Runs typically take about 1-2 hours to complete. Results consist of Amplification plots (c) and Melting Temperature curves (d). Final analysis consists of looking at what cycle fluorescence appeared and what the melting curve of each test looks like in comparison to the positive and negative control.

Figure 7-16 Interpreting RT-PCR

Well Sample Name Task Cycle threshold Melting temperature (Tm)
A1 positive control Standard 17.7 87.5
A2 negative control NTC 27.24 70.5
B1 Test 1 Unknown 19.91 87.5
B2 Test 2 Unknown 18.49 87.5
B3 Test 3 Unknown 14.65 87.5
B4 Test 4 Unknown 15.38 87.5
B5 Test 5 Unknown 31.21 67.1
B6 Test 6 Unknown 19.03 87.5
B7 Test 7 Unknown 21.3 87.9
B8 Test 8 Unknown 15.78 87.5
B9 Test 9 Unknown 22.48 72.3
B10 Test 10 Unknown 12.84 87.9
B11 Test 11 Unknown 14.51 87.2
B12 Test 12 Unknown 16.16 87.2
C1 Test 13 Unknown 25.17 65.2
C2 Test 14 Unknown 13.31 87.5

The table was generated by adding PCR primers specific for E. coli and running a RT-PCR reaction. Cycle threshold indicates the cycle at which the fluorescence detectedby the instrument passed a predetermined threshold, indicating the presence of a PCR product. Melting temperature is the temperature at which the amplified DNA double helix denatured into single strands. This is sequence specific. Any test PCR reaction should have approximately the same melting point as the positive control. Given this table of results, determine which of the 14 samples actually contained E. coli.

For more information on identifying and classifying bacteria, read the chapter on bacterial classification.

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