Detection of pork adulteration in beef with ladder-shape melting temperature isothermal amplification (LMTIA) assay

ABSTRACT The ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed technology, and the objective of this study is to establish the LMTIA assay for detection of pork in beef. The LMTIA primers were designed with the prolactin receptor gene of Sus scrofa as the target. The LMTIA reaction system was optimized, and the performance of the LMTIA assay in specificity, sensitivity and detection limit (LOD) was determined. Our results showed that the LMTIA assay was able to specifically detect 10 ng genomic DNA (gDNAs) of Sus scrofa, the sensitivity of the LMTIA assay was 1 ng genomic DNA of Sus scrofa, and the LMTIA assay was able to detect the pork adulterated in beef with 0.1% LOD, which was the same as that of reported PCR assay or LAMP assay. This study holds a promise for facilitating the surveillance of the commercial adulteration of pork in beef.


Introduction
High-value foods have recently become the objectives of food fraud, the main fraud ways include origin mislabeling and species adulteration (Brigante et al., 2020;Francois et al., 2020;Yang et al., 2020), for example, pork has been commonly adulterated into beef (Fengou et al., 2021), and the research on food authentication has already become the hot spot. The advanced technologies including nuclear magnetic resonance spectroscopy (Gallo et al., 2020), isotopic mass spectrometry (Martinelli et al., 2020), high resolution mass spectrometry , immunoassay (Mandli et al., 2018;Seddaoui & Amine, 2021), infrared spectroscopy (Ling et al., 2018;Rady & Adedeji, 2018), electrochemical genosensor (Flauzino et al., 2022), chemometrics (Song et al., 2021), nucleic acid detection have been studied for detection of food adulteration, among which the technologies other than DNA-based methods have disadvantages of low specificity (for example near infrared spectroscopy and immunoassay), unreliable results (for example electronic noses), high detection limit (for example electronic tongue, 11% LOD), expensive equipment (for example nuclear magnetic resonance spectroscopy, isotopic mass spectrometry and high resolution mass spectrometry) and so on (Zia et al., 2020), while the nucleic acid detection is suitable for species identification for its specification, sensitivity and accuracy (Amaral, 2021).
Ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed nucleic acids amplification technique. In the LMTIA, single-strand DNA template supply was used via the melting temperature (Tm) difference between the primer and its target sequence rather than thermal denaturation or enzyme catalysis. The melting temperature curve of a target sequence in the LMTIA reaction is ladder-shaped. The whole reaction is divided into the initial structure formation stage and the exponential amplification stage. The initial structure formation stage is composed of the cycle reactions of annealing, strand displacement, and synthesis. The exponential amplification stage is the length doubling process composed of the cycle reactions of annealing, strand displacement and synthesis, and strand displacement (Wang et al., 2021a).This technique has the following advantages over the previously established nucleic acids amplification techniques: simple mechanism, easy primer design, quick turn-around time, high specificity, high sensitivity, short target sequence, and wide application range of single-stranded DNA, and double-stranded DNA or RNA (for reverse transcriptase activity of Bst DNA Polymerase) as its target (Wang et al., 2021a). Thus, the objective of this study was to develop the LMTIA assay for detection of pork in beef, which may provide a promising solution for facilitating the surveillance of the commercial adulteration of pork in beef.

Target sequence selection and LMTIA primer design
According to the principle for the target sequence selection of LMTIA, the sequence with ladder-shaped melting temperature curve was selected as a target from the prolactin receptor (PRLR) gene of Sus scrofa (GenBank accession No.: DQ453511.1) using the software Oligo 7 (Molecular Biology Insights, Inc. Colorado Springs, CO, USA). The length of selected target was 96 nt, and its GC content was 56.2%. The melting temperature curve of the selected sequence is shown in Figure 1, and the 96-nt sequence was blasted in Genbank, only the DNA sequence from Sus scrofa completely hit, so the selected sequence was highly specific to pork (Sus scrofa) rather than to beef (taurus), which held the promise for detection of pork adulteration in beef. The LMTIA primers were designed with the online software Primer3Plus (http:// www.primer3plus.com/), and a 96-nt sequence was used as a target. The parameters of primers were set as described in previous report (Wang et al., 2021a). The primer sequences are listed in Table 1.

DNA preparation
The meat samples of Sus scrofa, Gallus gallus, Anas platyrhynchos, Felis catus, Canis lupus familiaris, Bos taurus and Capra hircus, which were the common commercial meat species on market especially in China, were provided by Henan Tuodao Biotechnology Co., Ltd, the genomic DNAs (gDNAs) of these samples were extracted using Animal Tissue DNA Extraction Kit (Tiangen Biotech (Beijing) Co., Ltd, Beijing, China) followed by the manufacturer's protocol. The gDNAs would be used for temperature optimization, specificity determination and sensitivity determination of the LMTIA assay.
Pork and beef meats were minced, 1.0 g, 10.0 g, 50.0 g 100.0 g and 200.0 g minced pork were mixed with 999.0 g, 990.0 g, 950.0 g, 900.0 g and 800.0 g minced beef, respectively, the samples of beef adulterated with 0.1%, 1%, 5%, 10% and 20% pork were prepared, the gDNAs were extracted from these samples using Animal Tissue DNA Extraction Kit (Tiangen Biotech (Beijing) Co., Ltd, Beijing, China), and the gDNAs of artificially prepared samples were for the detection limit determination of the LMTIA assay.

Specificity determination of the LMTIA assay
The specificity of the newly developed LMTIA assay was determined using 10 ng gDNAs of Sus scrofa, Gallus gallus, Anas platyrhynchos, Felis catus, Canis lupus familiaris, Bos taurus and Capra hircus, and the reaction mixtures were heated at the optimized temperature in a StepOne ™ System Real-Time PCR System (Applied Biosystems, CA, USA). This system was also used to collect fluorescent signals with 1.5 min intervals, and 40 signals were collected.

Sensitivity determination of the LMTIA assay for the Sus scrofa genomic DNA
The sensitivity of the newly developed LMTIA assay was determined using the gDNAs of Sus scrofa ranging from 10 ng to 10 fg, and the reaction mixtures were heated at the selected temperature in a StepOne ™ System Real-Time PCR System (Applied Biosystems, CA, USA). This system was also used to collect fluorescent signals with 1.5 min intervals, and 40 signals were collected.

Detection limit determination of the LMTIA assay for pork adulterated in beef
The detection limit (LOD) of the newly developed LMTIA assay was determined using the gDNAs extracted from the beef, which were adulterated with 0.1%, 1%, 5%, 10% and 20% pork, and the reaction mixtures were heated at the optimized temperature in an Archimed time resolution realtime fluorescence quantitative PCR system (RocGene (Beijing) Technology Co., Ltd, Beijing, China). This system was also used to collect fluorescent signals with 1.5 min intervals, and 40 signals were collected.

Selection of the LMTIA reaction temperature
As shown in Figure 2, in the LMTIA reaction, the results of all positive controls (with 10 ng gDNAs of Sus scrofa as template) were positive and the results of all negative controls (DNA template substituted by ddH 2 O) were negative PRLR gene of Sus scrofa at 57°C, 58°C, 59°C and 60°C. The highest amplification efficiency of the LMTIA reaction was observed at 60°C, although the amplification efficiency at 59°C was slightly lower than that at 60°C, the repeatability at 59°C was the better, therefore, and the temperature 59°C was selected as the optimal temperature of the LMTIA assay.

LMTIA is highly specific to pork
The specificity of LMTIA assay was tested using 10 ng gDNAs of Sus scrofa, Gallus gallus, Anas platyrhynchos, Felis catus, Canis lupus familiaris, Bos taurus and Capra hircus. As shown in Figure 3

Primer
Sequence (5′-3′) P GGCATCCCAGGGCGCTTTTTGTCTTGCCTTGTGTCTTTAACA D TGCCAAGACTTCCCCCTTTTGTCCTCGCAGTCGGAAGTG interleukin 2 (IL2) gene  had been previously selected for target sequence, while the 96 nt sequence on the prolactin receptor gene of Sus scrofa (GenBank accession No. DQ453511.1) was selected as target of the developed LMTIA assay, the sequence was highly specific to Sus scrofa, and it was also shown by the specificity assay that the developed LMTIA assay was highly specific to pork.

Sensitivity of the LMTIA assay for the Sus scrofa gDnas
Sensitivity of the LMTIA assay was tested using the gDNAs of Sus scrofa ranging from 10 ng to 10 fg, and the sensitivity was found to be 1 ng gDNAs of Sus scrofa, as shown in Figure 4. Moreover, the amplification of 1 ng gDNAs of Sus scrofa had entered into exponential stage within 20 cycles, the reaction time of LMTIA can be shorted to 30 min.

Detection limit of the LMTIA assay for pork adulterated in beef
The gDNAs extracted from the artificial samples of beef adulterated with 0.1%, 1%, 5%, 10% and 20% pork were used for the detection limit (LOD) determination of the LMTIA assay. As shown in Figure 5, the LOD of the LMTIA assay was 0.1%. Similarly, it was approved by the LOD determination that the reaction time of LMTIA can be shorted to 30 min.

Discussion
The LMTIA technique is the recently developed nucleic acid isothermal amplification upon modification of LAMP (Wang et al., 2021a). The isothermal amplification of PCR reaction was used in the technique via Tm difference between primers and their target sequences. It shortens the reaction time from more than 1 hr to less than 25 min using the primer design method of LAMP as reference, which has a clear single-strand template generation mechanism, high specificity, and sensitivity and is rapid and suitable for short target sequence amplification over LAMP. In this study, the LMTIA primers were designed with a 96 nt sequence on the prolactin receptor gene of Sus scrofa as a target, the LMTIA assay for detection of pork adulterated in beef was established. As shown by the experiments, the technique of LMTIA is reproducible.    In this study, the 96 nt sequence on the prolactin receptor gene of Sus scrofa (GenBank accession No.: DQ453511.1) has been selected as the target sequence of the LMTIA assay, the alignment in GenBank indicates that the target sequence is of high specificity to Sus scrofa, and it is also confirmed by the specificity determination that the developed LMTIA assay is highly specific to pork.
The developed LMTIA assay is able to detect the pork adulterated in beef with 0.1% LOD, while the LOD of electrochemical genosensor based on graphene acid is 9% (w/w) (Flauzino et al., 2022), the LOD of recombinase polymerase amplification (RPA) is 1% (w/w) (Cao et al., 2018), the LOD of the direct lysismultiplex PCR assay is 0.1% (w/w) (Zhao et al., 2021), and the LOD of LAMP is 0.1% (w/w) (Sul et al., 2019), so the LOD of the developed LMTIA is the same as previously reported nucleic acids amplification methods. The research has provided a new sensitive and rapid method for detection of pork adulterated in beef.
For the LMTIA assay, the Bst DNA polymerase (large fragment) should be modified to improve the catalytic efficiency and shorten the amplification time to less than 10 min, and the Bst DNA polymerase (large fragment) should be modified compatible with the UNG-dUTP system to reduce the product pollution. Moreover, the fluorescent probe suitable for the LMTIA technique should be designed to overcome the shortcomings of absence of internal amplification controls (IACs) as well as limitations of quantitative assessments and multiplexing (Kokkinos et al., 2014).

Conclusion
The LMTIA assay was developed to specifically detect pork adulterated in beef in this study, the sensitivity was 1 ng gDNAs of Sus scrofa, the LMTIA assay can detect 0.1% pork adulterated in beef, the experiment verified that the technique of LMTIA is reproducible and provided the a reference for authentication of other species meat using LMTIA technique.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by the Natural Science Foundation of China [Grant No. 31701689, 32172300], the Technology Project of Henan Province [Grant No. 212102110214] as well as Xuchang University and Local Government Cooperation Project.

Data availability statement
The data that support this study are available from the corresponding author upon reasonable request.