Rapid Detection of Listeria monocytogenes

in Raw Milk with Loop-Mediated Isothermal

Amplification and Chemosensor
Deguo Wang, Gaiping Zhang, Chengping Lu, Ruiguang Deng, Aimin Zhi, Junqing Guo, Dong Zhao, and Zhihong Xu

Abstract: Loop-mediated isothermal amplification (LAMP) allows a rapid amplification of nucleic acids under isothermal
conditions. It can be combined with a rhodamine-based dual chemosensor for much more efficient, field-friendly
detection of Listeria monocytogenes. In this report, LAMP was performed at 63 ◦ C for 10 min, followed by a rapid reaction
of DNA amplification and the byproduct, pyrophosphate ion, with a rhodamine-based dual chemosensor and Cu2+ is
visualized as a disappearance of red color. The detection limit of L. monocytogenes by the LAMP-chemosensor was 8 to
10 cells per reaction tube, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min.
Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving
100% concordance with the ISO 10560 reference method. The results showed that the LAMP-chemosensor method has
the advantages of better sensitivity and speed and less dependence on equipment than the standard Polymerase Chain
Reaction for specifically detecting low levels of L. monocytogenes DNA, and this can be useful in the field as a routine
diagnostic tool.
Keywords: Listeria monocytogenes, loop-mediated isothermal amplification (LAMP), rhodamine-based dual chemosensor

M: Food Microbiology
& Safety
Practical Application: The LAMP-chemosensor method reported here provided a powerful tool for detection of
L. monocytogenes in raw milk samples due to its specificity, sensitivity, and rapidity.

Introduction antibiotics, phenolic compounds, enzymes, polysaccharides, fats,

Listeria monocytogenes is a gram-positive bacterium and can cause
abortion, encephalitis, and septicaemia in humans and animals
(Kalorey and others 2008). In food safety control, detection
and monitoring of L. monocytogenes is an important microbio-
logical analysis. Although many methods for detecting L. mono-
cytogenes have been developed, the standard culture method is
commonly used. However, the disadvantages of the standard
method are that it is laborious and time consuming, requiring
nonselective pre-enrichment, selective enrichment, plating on dif-
ferential agar media, presumptive biochemical identification, and
serological confirmation. Impedance-based detection systems, im-
munological methods, and polymerase chain reaction have been
used to accelerate L. monocytogenes detection. 102 CFU/0.5 mL of
L. monocytogenes in milk was detected by immunomagnetic separa-
tion and real-time Polymerase Chain Reaction (PCR) (Yang and
others 2007). PCR for analyzing pure microbial cultures was very
effective, but limited by the presence of PCR inhibitors in the
analysis of real biological samples and food samples. The various
inhibitors, such organic and inorganic substances as detergents,
MS 20110475 Submitted 4/13/2011, Accepted 8/4/2011. Authors Wang and
Xu are with Chemistry and Chemical Industry College, Xuchang Univ., Xuchang
461000, China. Authors Wang, Zhang, Deng, Zhi, Guo, and Zhao are with Henan
Key Laboratory of Animal Immunology, Henan Academy of Agriculture Sciences,
Zhengzhou 450002, China. Author Lu is with Animal Medial College of Nanjing
Agricultural Univ., Nanjing, 210095, China. Direct inquiries to author Zhang
(E-mail: zhanggaiping2003@yahoo.com.cn).
proteins, and salts, reduced or inhibited the amplification effi-
ciency, resulting in the reduction of the detection sensitivity or
the production of false-negative results. Therefore, it is necessary
to develop a rapid, sensitive, and cost-effective method for micro-
biological examination of raw milk.
Recently, a new technique called loop-mediated isothermal am-
plification (LAMP) has been developed which can amplify nucleic
acids with high specificity, sensitivity, and rapidity under isother-
mal conditions (Notomi and others 2000). The method is easy
to perform (Nagamine and others 2002a), and is based on the
principle of the reaction performed by a DNA polymerase with
strand displacement activity and a set of 2 specially designed in-
ner primers (FIP and BIP) and 2 outer primers (F3 and B3).
LAMP is highly specific for the target sequence because 6 in-
dependent sequences (F1c, F2, F3, B1c, B2, and B3) recognized
the target sequence in the initial stage and 4 independent se-
quences (F1c, F2, B1c, and B2) amplified the target sequence
in the later stage of the LAMP reaction. Under an isothermal
condition, the amplification efficiency of the LAMP method is
extremely high because of the absence of a ramp time for ther-
mal change, because it is an isothermal reaction. Therefore, the
LAMP assay has the advantage of specificity, selectivity, and ra-
pidity over other nucleic acid amplification methods. Moreover,
Nagamine and others (2002b) advanced the method by putting
forward loop primers (LF and LB) that accelerated the LAMP
reaction.
Analysis of LAMP products was usually carried out by agarose
gel electrophoresis, followed by ethidium bromide staining. As a


C 2011 Institute of Food Technologists
R

doi: 10.1111/j.1750-3841.2011.02383.x Vol. 76, Nr. 9, 2011 r Journal of Food Science M611
Further reproduction without permission is prohibited
 Detection of Listeria monocytogenes . . .

used for determining the specification of LAMP. LAMP was per-

result, there was a carcinogenic hazard, and false positive results
were inevitable due to cross contamination. To help overcome formed in a total 25 μL reaction mixture containing 0.8 mM each
of FIP and BIP, 0.2 mM each of the outer primers, 0.4 mM each
these problems as well as to further simplify and speed up the total
of LF and LB, 1.6 mM dNTPs, 0.8 M betaine (Sigma-Aldrich,
time for the LAMP-based assay, a new analysis method was devel-
oped for detection of L. monocytogenes in raw milk with LAMP.St. Louis, Mo., U.S.A.), 20 mM Tris–HCl (pH 8.8), 10 mM KCl,
10 mM (NH4 )2 SO4 , 4 mM MgSO4 , 0.1% Triton X-100, 8 units
Materials and Methods of the Bst DNA polymerase (New England Biolabs, Beverly, Mass.,
U.S.A.), and 2 μL of extracted DNA, and LAMP was carried out
Synthesis of rhodamine-based dual chemosensor at 63 ◦ C for 40 min and terminated at 80 ◦ C for 3 min (Itano and
Rhodamine-based dual chemosensor was synthesized according others 2006). LAMP products were subjected to electrophoresis
to Huo and others (2010). on a 2.0% agarose gel, and the smallest amplified products were
sequenced by Shanghai Bioengineering Co. Ltd.
Binding property of rhodamine-based dual chemosensor
with pyrophosphate ion Detection limits of LAMP and PCR for artificial
Absorption spectra of the rhodamine-based dual chemosensor contaminated raw milk
with Cu2+ and various concentrations of pyrophosphate ion in The DNA extracted from artificially seeded raw milk samples
methanol were recorded with a Techcomp UV-8500 spectropho- was used as templates for determining detection limits of LAMP
tometer (Shanghai, China), and the wavelength used was from and PCR. LAMP was performed with the same amplification
400 to 600 nm. system and conditions as mentioned above. The amplified prod-
ucts were subjected to electrophoresis on a 2.0% agarose gel and
Artificial contamination of raw milk visualized under UV light of ChemiDocXRS (Bio-Rad Labora-
Food samples for artificial contamination were tested according tories, Inc., Benicia, Calif., U.S.A.) after ethidium bromide stain-
to ISO 10560 to ensure that they were L. monocytogenes nega-
tive prior to inoculation (Kaclı́ková and others 2003). The raw
M: Food Microbiology

Table 1–Primers for the isothermal amplification of L. monocytogenes

milk free of L. monocytogenes was artificially contaminated with DNA.
L. monocytogenes CMCC54002 as reference strain according to
& Safety

Peter and others (2006), and live L. monocytogenes in the naturally Primer Sequence
contaminated raw milk samples was confirmed with PALCAM F3 5 -CTGGTTACACTAAATATGTATTTGC-3
Agar. B3 5 -GCCGTGGATGTTATCGTAT-3

FIP 5 -AGGTTTTGCTTGAGATTCAGAGATtttt
TAATCTCCCTTCCACGTACT-3
DNA extraction BIP 5 -TTGACTATGGTAGCGGAATTTCTCtttt
Ten-fold dilution of 25 mL artificially contaminated raw milk TTAACGCCATTGTCTTGC-3
was carried out in a sterile buffer (20 mM Tris–HCl, pH 8.0, LF 5 -GTAGTGCTAGCGTATTGTGCG-3
LB 5 -GGTATCTACGTTGGTAATGGTCAA-3
2 mM EDTA, pH 8.0, and 1.2% Triton X-100), and then the
DNA of the dilution series was extracted according to the pub-
lished method (Yano and others 2007), One milliliter of each Table 2–Bacterial strains used in this study and their sources.
diluted sample was centrifuged for 1 min at 10000 × g. The su- Strains Sources
pernatant was removed. The pellets were suspended in 200 μL
Listeria monocytogenes Offered by CMCCa
of buffer (20 mM Tris–HCl, pH 8.0, 2 mM EDTA, pH 8.0, and
CMCC54002
1.2% Triton X-100), and the tubes were incubated at 96 ◦ C for Listeria monocytogenes Offered by Heilongjiang Import and Export
15 to 30 min; after vortex mixing again, the tubes were subjected HJ-35 Inspection and Quarantine Bureau
to centrifugation at 10000 × g for 2 to 3 min, and the supernatant Salmonella sp CGMCC Offered by CGMCCb
was used for determining sensitivities of the LAMP method and 1.1552
Salmonella enterica Deposited in DICCc
PCR assay. At the same time, the precise number of CFU in ATCC13076
the dilution series was obtained by the plate count method using ETEC: 44247(6:15:16) Offered by CMCC
tryptone soy agar with 0.6% yeast extract. EPEC: 44706(111:58:-) Offered by CMCC
Pseudomonas fluorescence Offered by CGMCC
CGMCC 1.1802
Primer design Pseudomonas putida Offered by CGMCC
Based on the iap gene (GenBank Accession NC_003210) of CGMCC1.1819
L. monocytogenes EGD-e, 6 primers were designed using Primer- Staphylococcus aureus Offered by Heilongjiang Province Applied
Explorer 3 (Table 1). ATCC25923 Microbiology Research Institute
Staphylococcus aureus HJ-02 Offered by Heilongjiang Import and Export
Inspection and Quarantine Bureau
Specification of the LAMP method Shigella spp HJ-14 Offered by Heilongjiang Import and Export
Two strains of L. monocytogenes and 12 non-L. monocytogenes Inspection and Quarantine Bureau
Escherichia coli Offered by Heilongjiang Province Applied
strains were used for the specificity study (Table 2). L. monocy- ATCC25922 Microbiology Research Institute
togenes were cultured overnight at 37 ◦ C in tryptone soy broth Escherichia coli O157:H7 Offered by Heilongjiang Import and Export
with 0.6% yeast extract (Rossmanith and others 2006) and the Inspection and Quarantine Bureau
others in Luria-Bertani (LB) broth. DNA from these pure cul- Bacillus cereus KLDS71 Deposited in DICC
tures was extracted according to the manufacturer’s instructions a
b
CMCC: National Center for Medical Culture Collection Center.
CGMCC: China General Microbiological Culture Collection Center.
of a UNIQ10 Column DNA Extraction Kit (Shanghai Bioengi- c
DICC: Dairy Industry Culture Collection Center of Key Laboratory of Dairy Science,
neering Co., Ltd., Shanghai, China), and the DNA template was Ministry of Education of China.

M612 Journal of Food Science r Vol. 76, Nr. 9, 2011

Detection of Listeria monocytogenes . . .

ing (Yano and others 2007). And the PCR reaction was carried Sensitivity of the LAMP method and PCR assay
out according to the method described by Kwiatek and others Comparative analysis of sensitivity of L. monocytogenes detec-
(2003). tion by LAMP and PCR was carried out using a dilution series
of artificially contaminated raw milk. The detection limit of the
Sensitivity of electrophoresis and rhodamine-based dual LAMP assay was found to be 186 CFU/mL which corresponds
chemosensor to 8 to 10 cells per reaction tube, while the detection limit of
LAMP was carried out at 65 ◦ C for 10, 15, 20, 25, 30, 35, the PCR was 1.86 × 105 CFU/mL, it was indicated that LAMP
40, 45, and 50 min, respectively, and then terminated at 80 ◦ C was 103 -fold more sensitive than conventional PCR (Figure 3).
for 3 min. After LAMP products were analyzed by agarose gel The sensitivity of the LAMP assay was consistent with reports
electrophoresis, 5 μL red rhodamine-based dual chemosensor was concerning Salmonella spp, Escherichia coli, Flavobacterium columnare,
added to the residual amplified liquid. ammonia-oxidizing bacteria, and Escherichia coli O157 (Yano and
others 2007; Hara-Kudo and others. 2008; Okamura and others.
L. monocytogenes determination of naturally 2008; Wang and others 2008; Wang and others 2009), but the de-
contaminated raw milk tection limit of the PCR method was higher than that reported for
A total of 125 raw milk samples analyzed in the study were tested L. monocytogenes (Rossmanith and others 2006), which may be due
for L. monocytogenes contamination by the LAMP-chemosensor to the wide range of inhibitors in template DNA extracted from
method without enrichment and after being enriched in tryptone artificially contaminated raw milk (Kaneko and others 2007).
soy broth with 0.6% yeast extract for 6 h at 37 ◦ C, respectively,
and by the ISO 10560 International reference method (Kaclı́ková
Sensitivity of electrophoresis and rhodamine-based dual
and others 2003).
chemosensor
Results and Discussion As Fig. 4 indicates, the amplified products cannot be detected
by agarose gel electrophoresis when LAMP was performed for less
Absorption property of rhodamine-based dual than 25 min; however, they can be detected by rhodamine-based
chemosensor with pyrophosphate ion dual chemosensor via color change when LAMP was carried out

M: Food Microbiology
The absorption spectra of rhodamine-based dual chemosensor for 15 min, so the total assay time with rhodamine-based dual
(10 μM) with Cu2+ (10 μM) and various concentration of py- chemosensor including 10 min for rapid DNA extraction was

rophosphate ion in methanol are shown in Figure 1, indicating
that the absorption increased with decreasing concentration of
pyrophosphate. The color of rhodamine-based dual chemosen-
sor changed from red to colorless on the addition of 1.0 μM
pyrophosphate ion, a change which can be detected by the naked
eye. The analytical detection limit for pyrophosphate ion by the
naked eye was as low as 1.0 μM.
Specification of LAMP assay
As shown in Figure 2, the primers only amplified the sequences
from 2 stains of L. monocytogenes, but not from other bacterial
species and negative control. The sequencing results indicated that
the amplified products length of 2 stains of L. monocytogenes was
223 bp. The sequences were 100% concordance with iap gene of
L. monocytogenes EGD-e.
& Safety
approximately 30 min.
In LAMP, a large amount of DNA was synthesized, yielding a
large amount of the pyrophosphate ion as a by-product. The reac-
tion of pyrophosphate ion with rhodamine-based dual chemosen-
sor and Cu2+ results in the disappearance of the red colour, which
enabled visual discrimination of results, and gives the rhodamine-
based dual chemosensor method advantages over agarose gel elec-
trophoresis (Wang and others 2010), turbidity (Seki and others
2005), SYBR GREEBI (Bipin and others 2007), calcein (Tomita
and others 2008), and lateral flow dipstick (Teeranart and others
2010). Agarose gel electrophoresis has a carcinogenic hazard and

Figure 2–Specificity of the LAMP reaction for detection of iap gene.

M, Marker DL2000; Lane 1, L. monocytogenes CMCC54002; Lane 2,
L. monocytogenes HJ-35; Lane 3, Salmonella sp CGMCC 1.1552; Lane 4,
Salmonella enterica ATCC13076; Lane 5, ETEC: 44247(6:15:16); Lane 6,
EPEC: 44706(111:58:-): Lane 7, Pseudomonas fluorescence CGMCC 1.1802;
Lane 8, Pseudomonas putida CGMCC1.1819; Lane 9, Staphylococcus aureus
Figure 1–Absorption spectra of rhodamine-based dual chemosensor ATCC25923; Lane 10, Staphylococcus aureus HJ-02; Lane 11, Shigella spp
(10 μM) and Cu2+ (10 μM) in methanol in the presence of different con- HJ-14; Lane 12, Escherichia coli ATCC25922; Lane 13, Escherichia coli
centrations of P2 O7 4− . O157:H7; Lane 14, Bacillus cereus KLDS71; N, negative contrast.

Vol. 76, Nr. 9, 2011 r Journal of Food Science M613

 Detection of Listeria monocytogenes . . .

can be confused easily by cross contamination; determination of by the ISO 10560 method. However, when the raw milk samples
turbidity needs expensive instrumentation – Loopamp real-time were enriched for 6 h at 37 ◦ C, the number of positive samples
turbidimeter; SYBR GREEBI cannot discriminate between am- detected by the LAMP-chemosensor method was the same as that
plified DNA and primer dimers, which frequently resulted in a by the ISO 10560 method, as shown in Table 3.
false positive result (Daniel and others 2007); visual analysis with
calcein was performed under a fluorescent lamp, and the lateral
Table 3– L. monocytogenes determination of naturally contaminated
flow dipstick is based on both labeled primer and probe, which raw milk.
increases test cost and perplexed detection process.
LAMP- LAMP-
chemosensor chemosensor
L. monocytogenes detection of naturally contaminated without with
raw milk Number ISO10560a enrichment enrichment
of
A total of 125 raw milk samples were collected from Xiangfang samples Positive Negative Positive Negative Positive Negative
Farm of Northeast Agricultural University. Of 125 intact samples,
10 were tested by the LAMP-chemosensor method contained 125 12 113 10 115 12 113
a
L. monocytogenes, while 12 of the 125 samples were tested positive Raw milk sample testing by ISO10560 was performed in duplicate.
M: Food Microbiology
& Safety

Figure 3–Sensitivity comparison of LAMP and PCR for detection of L. monocytogenes CMCC54002 in raw milk. M, Marker 2000; N, negative control;
Lanes 1–8, LAMP carried out using the dilution series of artificially contaminated raw milk, 10−1 , 10−2 , 10−3 , 10−4 , 10−5 , 10−6 , 10−7 , 10−8 , respectively;
Lanes 9–16, PCR carried out using the dilution series of artificially contaminated raw milk, 10−1 , 10−2 , 10−3 , 10−4 , 10−5 , 10−6 , 10−7 , 10−8 , respectively.
All the products were electrophoresed on 2% agarose gels and stained with ethidium bromide.

Figure 4–Sensitivity of electrophoresis and rhodamine-based dual chemosensor. LAMP was carried out at 65 ◦ C for 10, 15, 20, 25, 30, 35, 40, 45, and
50 min, which corresponded to Lanes 1–9, respectively; the first tube was rhodamine-based dual chemosensor solution.

M614 Journal of Food Science r Vol. 76, Nr. 9, 2011

Detection of Listeria monocytogenes . . .
Conclusion
The results indicated that the LAMP-chemosensor method
had the advantages of enhanced sensitivity and speed and less
dependence on equipment than the standard PCR method for
specifically detecting low levels of L. monocytogenes DNA, which
should be helpful in basic research on food safety, medicine
and pharmacy, environmental hygiene, point-of-care testing,
and more.
Acknowledgments
This study was financially supported by Key Science and
Technology Project of Henan Province (112102310370) and Sci-
entific Research Project of Xuchang Univ. (2011B026).
References
Bipin RB, Chandra I, Robert MW, Pradip M, Parmjeet SR, Gaurav G, Meena A, Rakhi T,
Gina G, Abhay V. 2007. Development of a loop-mediated isothermal amplification assay for
rapid detection of BK virus. J Clin Microbiol 45:1581–7.
Daniel HP, Mallika I, Abul MF, Mahtabuddin H, Emran BY, Kamolrat S, Sue JL., Nicholas PJD,
Arjen MD. 2007. Loop-mediated isothermal PCR (LAMP) for the diagnosis of Falciparum
Malaria. Am J Trop Med Hyg 77:972–6.
Hara-Kudo Y, Konishi N, Ohtsuka K, Hiramatsu R, Hiroyuki T, Konuma H, Takatori K.
2008. Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating methods and
LAMP method: a collaborative study. Int J Food Microbiol 122:156–61.
Huo FJ, Su J, Sun YQ, Yin CX, Tong HB, Nie ZX. 2010. Rhodamine-based dual chemosensor
for the visual detection of copper and the ratiometric fluorescent detection of vanadium. Dyes
Pigm 86:50–5.
Itano T, Kawakami H, Kono T, Sakai M. 2006. Detection of fish nocardiosis by loop-mediated
isothermal amplification. J Appl Microbiol 1006:1381–7.
Kaclı́ková E, Pangallo D, Drahovská H, Oravcová K, Kuchta T. 2003. Detection of Listeria
monocytogenes in food, equivalent to EN ISO 11290-1 or ISO 10560, by a three-day polymerase
chain reaction-based method. Food Control 14:175–9.
Kalorey DR, Warke SR, Kurkure NV, Rawool DB, Barbuddhe SB. 2008. Listeria species in
bovine raw milk: a large survey of Central India. Food Control 19:109–12.
Kaneko H, Kawana T, Fukushima E, Suzutani T. 2007. Tolerance of loop-mediated isother-
mal amplification to a culture medium and biological substances. J Biochem Bioph Meth
70:499–501.
Kwiatek K, Wojdat E, Rzezutka A, Rola J, Rózycki M. 2003. Comparison of PCR and other
methods in the detection of Listeria monocytogenes in milk inoculated experimentally with the
bacteria. Bull Vet Inst Puawy 47:357–62.
Nagamine, K., Kuzuhara, Y., Notomi, T. 2002. Isolation of singles tranded DNA from
loop-mediated isothermal amplification products. Biochem Biophys Res Commun 290:
1195–8.
Nagamine K, Hase T, Notomi T. 2002. Accelerated reaction by loop-mediated isothermal
amplification using loop primers. Mol Cell Probes 16:223–9.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. 2000.
Loop-mediated isothermal amplification of DNA. Nucleic Acid Res 28:e63.
Okamura M, Ohba Y, Kikuchi S, Suzuki A, Tachizaki H, Takehara K, Ikedo M, Ko-
jima T, Nakamura M. 2008. Loop-mediated isothermal amplification for the rapid, sen-
sitive, and specific detection of the O9 group of Salmonella in chickens. Vet Microbiol
132:197–204.
Peter R, Martina K, Martin W, Ingeborg H. 2006. Detection of Listeria monocytogenes in food
using a combined enrichment/real-time PCR method targeting the prfA gene. Res Microbiol
157:763–71.
Rossmanith P, Krassnig M, Wagner M, Hein I. 2006. Detection of Listeria monocytogenes in
food using a combined enrichment/real-time PCR method targeting the prfA gene. Res in
Microbiol 157:763–71.
Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M. 2005. Loop-mediated isothermal
amplification method targeting the lytA gene for detection of Streptococcus pneumoniae. J Clin
Microbiol 43:1581–6.
Teeranart P, Saengchan S, Timothy WF, Wansika K. 2010. Rapid and sensitive detection of
Macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-
mediated isothermal amplification combined with a lateral flow dipstick. Mol Cell Probes
24:244–9.
Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated isothermal amplification (LAMP)
of gene sequences and simple visual detection of products. Nat Protoc 3:877–82.
Wang L, Li L, Alam MJ, Geng YH, Li ZY, Yamasaki S, Shi L. 2008. Specific and rapid detection
of foodborne Salmonella by loop-mediated isothermal amplification method. Food Res Int
41:69–74.
Wang DG, Liu, Huo GC, Ren DX, Li YG. 2009. Development and evaluation of a loop-
mediated isothermal amplification (LAMP) method for detecting Escherichia coli O157 in raw
milk. J Rapid Meth Atu Mic 17:55–6.
M: Food Microbiology
Wang DG, Huo GC, Ren DX, Li YG. 2010. Development and evaluation of a loop-mediated
isothermal amplification (LAMP) method for detecting Listeria monocytogenes in raw milk. J
Food Safety 30:251–62.
& Safety
Yang H, Qu LW, Wimbrow AN, Jiang XP, Sun YP. 2007. Rapid detection of Listeria mono-
cytogenes by nanoparticle-based immunomagnetic separation and real-time PCR. Int J Food
Microbiol 118:132–8.
Yano A, Ishimaru R, Hujikata R. 2007. Rapid and sensitive detection of heat-labile I and
heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal
amplification. J Microbiol Meth 68:414–20.
Vol. 76, Nr. 9, 2011 r Journal of Food Science M615