Chapter 28
PCR for Detection and Identification of Yersinia pestis
Yong Zhao
Abstract
Molecular methods aimed at Yersinia pestis detection and identification mainly rely on the polymerase chain
reaction (PCR) technique, including conventional PCR and real-time PCR. PCR-based assays are more
sensitive and rapid than traditional methods, as the isolation and growth of Y. pestis from specimens are
laborious and time-consuming. Y. pestis target gene sequences can also be quantified using real-time PCR.
In this chapter, methods to extract Y. pestis genomic DNA from simulated soil samples are described. PCR
and real-time PCR methods for sensitive and quantitative detection of Y. pestis are also discussed.
Key words Yersinia pestis, Molecular diagnosis, PCR assay
1 Introduction
The polymerase chain reaction (PCR) technique is an important
tool for detection and identification of Yersinia pestis, as it is very
sensitive and can be rapidly performed. PCR-based assays usually
target specific gene sequences of Y. pestis, such as caf1 [1], pla [2],
ymt [3], or hms [4] on the plasmids or the 3a sequence [5] on the
chromosome. The target gene sequence can be detected via gel
electrophoresis (conventional PCR) or fluorescence analysis (real-
time PCR). Compared with conventional PCR, real-time PCR can
simultaneously amplify and detect the DNA target in one tube,
avoiding contamination of the DNA product and providing greater
sensitivity and specificity. Essentially, two fluorescent chemistries
are commonly used in real-time PCR: SYBR Green and the hydro-
lysis probe (mostly employing the TaqMan probe [6]). The Taq-
Man probe is more expensive than SYBR Green, but it ensures
higher specificity because only the specific amplicon is measured
[7]. Table 1 lists primer sets and TaqMan probes used in real-time
PCR for Y. pestis detection [1, 3].
In this chapter, we describe a protocol to detect and identify
Y. pestis live attenuated strain EV76 in soil samples by using both
conventional and real-time PCR targeting the 3a sequence
Ruifu Yang (ed.), Yersinia Pestis Protocols, Springer Protocols Handbooks, https://doi.org/10.1007/978-981-10-7947-4_28,
© Springer Nature Singapore Pte Ltd. 2018
243
244 Yong Zhao
Table 1
Primers and probes used in real-time PCR for Y. pestis detection
Target gene Primers and probes Sequence (5–30 )
Pla Pla-forward primer GTAATAGGTTATAACCAGCGCTT
Pla-reverse primer AGACTTTGGCATTAGGTGTG
Pla-TaqMan probe Fam-ATGCCATATATTGGACTTGCAGGCCAGT-Tamara
caf1 caf1-forward primer AGGTAAACGGTGAGAACCTTGTG
caf1-reverse primer CAATTGAGCGAACAAAGAAATCC
caf1-TaqMan probe Fam-ATGACGTCGTCTTGGCTACGGGCA-Tamara
3aa 3a-forward primer GGACGGCATCACGATTCTCT
3a-reverse primer CCTGAAAACTTGGCAGCAGTT
3a-TaqMan probe Fam-AAACGCCCTCGAATCGCTGGC-Tamara
3a sequence-negative Y. pestis strains have been found in Shaanxi Province, China [8]
a
(GenBank accession no. AF350075) on the Y. pestis chromosome.
This protocol can be used to identify Y. pestis in the laboratory or in
the field for plague surveillance and biosafety screening.
2 Materials
2.1 Preparation of 1. Y. pestis live attenuated strain EV76.
Soil Samples Spiked 2. Luria-Bertani (LB) broth.
with Y. pestis
3. Brain-heart infusion (BHI) agar (BD BBL™).
4. Saline solution (0.9% NaCl).
5. Soil sample (collected from a yard or garden and air-dried at
room temperature).
2.2 DNA Extraction 1. Distilled water (dH2O).
from Simulated Soil 2. 1.5-mL Eppendorf tubes.
Samples
3. Vortex.
4. Centrifuge.
5. Water bath.
2.3 Conventional 1. 10 PCR buffer (50 mM KCl, 20 mM MgCl2, 200 μM
PCR with Gel dNTPs).
Electrophoresis 2. Taq DNA polymerase (5 U/μL).
3. Y. pestis 3a sequence forward and reverse primers (10 μM):
Forward primer: 50 -TGTAGCCGCTAA GCACTACCATCC-30
Reverse primer: 50 -GGCA ACAGCTCAACACCTTTGG-30
4. Negative control: nuclease-free water.
PCR for Detection and Identification of Yersinia pestis 245
5. Positive control: plasmid DNA samples containing the target
3a sequence.
6. PCR tubes or 96-well plates.
7. Thermal cycler (T100, Bio-Rad).
8. 1.5% agarose gel containing SYBR Green I (10,000, Solar-
bio-SY1020).
9. Molecular weight ladder (1200, 1000, 800, 400, 200, and
100 bp).
10. UV light.
2.4 Real-Time PCR 1. 2 LightCycler 480 Probes Master (Roche Diagnostics).
2. 3a sequence primers and probes:
3a-forward primer and 3a-reverse primer (10 μM) (see Table 1).
3a-TaqMan probe (5 mM) (see Table 1).
3. Nuclease-free water.
4. Standard samples: plasmid DNA samples containing the target
3a sequence. The concentration of the plasmid solution is
determined by a UV spectrophotometer to calculate copy
numbers of the target DNA. The plasmid solution is then
serially diluted by tenfold to prepare standard templates with
known copy numbers of target DNA.
5. Real-time PCR tubes or 96-well plates.
6. Real-time PCR thermal cycler (LightCycler 480 II, Roche).
3 Methods
3.1 Preparation of 1. Grow Y. pestis live attenuated strain EV76 in LB broth at 26 C
Soil Samples Spiked to stationary phase or mid-logarithmic growth phase.
with Y. pestis 2. Centrifuge bacteria at 5000g for 10 min. Wash bacterial cells
twice with saline solution.
3. Determine the number of viable cells by counting colony-
forming units (CFUs) on BHI agar plates.
4. Adjust the bacteria concentration to 2 108 CFU/mL, and
serially dilute the bacteria by tenfold with saline solution
(2 103–2 107 CFU/mL).
5. Add 100 μL of each bacterial dilution to 0.2 g soil samples.
6. Incubate spiked soil samples at 4 C overnight for complete
adsorption of the bacteria to the soil.
3.2 DNA Extraction 1. Add 1 mL dH2O to each spiked soil sample and vortex
from Simulated Soil thoroughly.
Samples
246 Yong Zhao
2. Centrifuge the mixture at 1000g for 1–2 min at room tem-
perature. Then transfer the supernatant to an Eppendorf tube.
3. Repeat Steps 1 and 2 once to collect as many bacteria as
possible.
4. Centrifuge the supernatant at 5000g for 10 min. Then add
100 μL dH2O to the pellet.
5. Incubate the collected bacteria in a boiling water bath for
10 min.
6. Spin down the solution at 5000g for 10 min. Transfer the
supernatant to another Eppendorf tube. The solution can be
used as DNA template for PCR.
3.3 Conventional 1. Prepare the PCR mix by adding the following per reaction
PCR with Gel (23 μL total volume) (see Notes 1 and 2):
Electrophoresis 2.5 μL 10 PCR buffer (50 mM KCl, 20 mM MgCl2, 200 μM
dNTPs).
0.5 μL each of 10 μM forward and reverse primers.
0.2 μL Taq DNA polymerase (5 U/μL).
19.8 μL nuclease-free water.
Mix gently by pipetting up and down.
2. Add 5 μl template DNA (i.e., sample DNA, negative control
DNA, or positive control DNA dilution) to the corresponding
wells (see Note 3).
3. Perform the PCR reaction in the thermal cycler (T100,
Bio-Rad) using the following cycling conditions:
1 Cycle 3 min 94 C (initial denaturation)
30 Cycles 30 s 94 C (denaturation)
30 s 58 C (annealing)
1 min 72 C (extension)
1 Cycle 5 min 72 C (final extension)
4. Cool PCR products to 4 C either in the PCR machine or by
transferring to a refrigerator.
5. Prepare a 1.5% agarose gel containing SYBR Green I (10,000).
Electrophorese 5–10 μL PCR products along with a molecular
weight ladder at a voltage of 60–80 V/cm for 15–30 min.
6. Visualize PCR products under UV light.
7. Data analysis for conventional PCR: Positive samples contain-
ing Y. pestis 3a sequence will show a characteristic amplicon of
276 bp in length (Fig. 1).
PCR for Detection and Identification of Yersinia pestis 247
Fig. 1 PCR product analysis for Y. pestis detection using conventional PCR. Lane
M contains a molecular weight marker (1200, 1000, 800, 400, 200, and 100 bp);
lanes 1 and 2 contain negative controls; lane 3 contains the positive control;
lanes 4–8 show samples containing the expected 276 bp DNA product for
Y. pestis 3a sequence, corresponding to DNA samples extracted from soil
samples spiked with Y. pestis (2 103–2 107 CFU/mL)
3.4 Real-Time PCR 1. Prepare the real-time PCR mix by adding the following per
reaction (20 μL total volume) (see Note 4):
10.0 μL 2 LightCycler 480 Probes Master (Roche
Diagnostics).
1.0 μL each of 10 μM forward and reverse primers.
1.0 μL of TaqMan probe (5 mM).
8.0 μL nuclease-free water.
Mix gently by pipetting up and down.
2. Add 5 μL uncharacteristic DNA templates or water (to serve as
a negative control) to appropriate real-time PCR tubes or plates
containing 20 μL of real-time PCR mix. To obtain a standard
curve for Y. pestis quantification, run five PCRs with standard
samples containing the 3a sequence (10–108 copies/μL) as
templates in parallel with the uncharacteristic DNA samples
(see Note 5).
3. Perform real-time PCR in the LightCycler 480 II thermal
cycler using the following cycling parameters (see Note 6):
1 cycle 5 min 95 C (pre-denaturation)
45 cycles 5s 95 C (denaturation)
30 s 60 C (annealing and extension)
1 cycle 1 min 40 C (cool down)
4. Data analysis for real-time PCR
248 Yong Zhao
Fig. 2 Amplification curves (a) and the standard curve (b) for serially diluted standard samples (10–108 copies/
μL) in real-time PCR analysis. In b the x-axis represents the log10 concentrations of serially diluted template
(10–108 copies/μL), and the y-axis shows Ct values. The quantification equation is determined via Origin 8.0
or Microsoft Office Excel. The coefficient of determination (R2) implies the linearity of the standard curve
(a) Amplification curves and the cycle threshold (Ct) of each
DNA sample are obtained by using the LightCycler soft-
ware program (version 4.0) with second derivative algo-
rithms (Fig. 2a). A reaction with a Ct value 39 is
considered to be positive, and a reaction with a Ct value
>39 is deemed to be negative (see Note 7).
(b) For Y. pestis quantification, plot the standard curve with
the DNA concentration of the standard samples as the
x-axis and Ct values as the y-axis (Fig. 2b).
(c) Quantify the concentration or amount of unknown DNA
samples by using the quantification equation obtained
above.
4 Notes
1. The following precautions must be noted when performing
PCR. (1) It is recommended to have two independent labora-
tories or room to physically separate the PCR mix assembly
(including DNA isolation and PCR setup) from post-
amplification analysis. (2) A dedicated set of pipet aids and
equipment must be used in setting up PCRs and always use
aerosol-barrier tips. (3) It is better to maintain all PCR reagents
at 4 C by submerging in a cold chamber or an ice bucket when
setting up PCRs. The same precautions should be followed
when analyzing samples by real-time PCR methods.
PCR for Detection and Identification of Yersinia pestis 249
2. Add one or two extra reactions to compensate for pipetting
error when preparing PCR master mix. For example, if prepar-
ing a master mix for ten reactions, add 12 times the suggested
volumes of each reagent.
3. The PCR experiment should include a positive control (DNA
or RNA template from known positives) and a negative control
(contains all reagents except DNA template).
4. It is critical to perform real-time PCR experiments in accor-
dance with the Minimum Information for Publication of
Quantitative Real-Time PCR Experiments (MIQE)
guidelines [9].
5. Standard samples to obtain the standard curve should be
included in every real-time PCR experiment to quantify
target DNA.
6. Signal acquisition mode is “single” at each cycle end of the
amplification step.
7. Negative controls should not exhibit fluorescence growth
curves that cross the Ct value. A false positive may indicate
sample contamination. Invalidate the run and repeat the reac-
tion under stricter conditions.
References
1. Qu S, Shi Q, Zhou L, Guo Z, Zhou D, Zhai J, rapid detection of Yersinia pestis. Appl Environ
Yang R (2010) Ambient stable quantitative PCR Microbiol 67(8):3759–3762
reagents for the detection of Yersinia pestis. 6. Holland PM, Abramson RD, Watson R, Gelfand
PLoS Negl Trop Dis 4(3):e629 DH (1991) Detection of specific polymerase
2. Loiez C, Herwegh S, Wallet F, Armand S, chain reaction product by utilizing the 5–30 exo-
Guinet F, Courcol RJ (2003) Detection of Yer- nuclease activity of Thermus aquaticus DNA
sinia pestis in sputum by real-time PCR. J Clin polymerase. Proc Natl Acad Sci U S A 88
Microbiol 41(10):4873–4875 (16):7276–7280
3. Riehm JM, Rahalison L, Scholz HC, Thoma B, 7. Liu H (2006) TaqMan probe array for quantita-
Pfeffer M, Razanakoto LM, Al Dahouk S, tive detection of DNA targets. Nucleic Acids Res
Neubauer H, Tomaso H (2011) Detection of 34(1):e4–e4
Yersinia pestis using real-time PCR in patients 8. Qi Z, Wu Y, Li Y, Li C, Yang X, Zhang Q, Xin Y,
with suspected bubonic plague. Mol Cell Probes Jin Y, Wei R, Cui Y (2015) 3a-negative Yersinia
25(1):8–12 pestis, China. Infec Dis Transl Med 1(2):61–62
4. Hinnebusch BJ, Perry RD, Schwan TG (1996) 9. Bustin SA, Benes V, Garson JA, Hellemans J,
Role of the Yersinia pestis hemin storage (hms) Huggett J, Kubista M, Mueller R, Nolan T,
locus in the transmission of plague by fleas. Sci- Pfaffl MW, Shipley GL et al (2009) The MIQE
ence 273(5273):367–370 guidelines: minimum information for publica-
5. Radnedge L, Gamez-Chin S, McCready PM, tion of quantitative real-time PCR experiments.
Worsham PL, Andersen GL (2001) Identifica- Clin Chem 55(4):611–622
tion of nucleotide sequences for the specific and
