Differentiation of deer tendons from cattle tendons by a loop-mediated isothermal amplification (LAMP) test and bone remodeling bioassays
- Li-Li Jiang†1, 2,
- Cheuk-Lun Liu†1, 2,
- Yuk-Lau Wong†1, 2,
- Chun-Fong Nip3 and
- Pang-Chui Shaw1, 2, 3Email author
© Jiang et al. 2015
Received: 6 March 2015
Accepted: 26 October 2015
Published: 12 November 2015
Deer tendons are believed more effective than cattle tendons in tonifying kidney yang (shen yang) and enhancing bone and tendons. This study aims to differentiate the two types of tendons by a loop-mediated isothermal amplification (LAMP) test and bone remodeling bioassays.
Internal control primers to detect both types of tendons and specific primers for deer tendons were designed according to a sequence analysis. The LAMP test was set up and the results were analyzed by conventional gel electrophoresis, real-time fluorescence observation, and colorimetric detection. Crude tendon extracts were prepared by water extraction to compare their effects on bone. The anti-osteoclastic effects were investigated on mouse pre-osteoclast Raw264.7 cells by cell viability determination and tartrate-resistant acid phosphatase staining. The osteogenic effects were examined using rat osteoblast-like UMR106 cells by evaluation of cell proliferation, alkaline phosphatase activity, and calcium deposition. The relative gene expressions of bone remodeling-related markers, including nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase, cathepsin K, and osteoprotegerin/receptor activator of NF-κB ligand, were determined by real-time PCR.
In the LAMP test, both deer and cattle tendons were detected in the control reactions, while only deer tendons were amplified by the specific LAMP test. In the bioassays, both tendons inhibited the viability and differentiation of pre-osteoclast Raw264.7 cells, and promoted the proliferation and mineralization of osteoblast-like UMR106 cells. The mRNA expressions of bone remodeling-related markers were consistent with the results of the bioassays.
This study demonstrated that the isothermal LAMP test can distinguish between deer tendons and cattle tendons. Both types of tendons exhibited similar beneficial effects on bone remodeling according to the bioassay findings.
In traditional Chinese medicine, deer tendons are the dried limb tendons of sika deer (Cervus nippon) or red deer (Cervus elaphus), while cattle tendons are the dried tendons of domestic cattle (Bos taurus). In East Asia, deer tendons are believed to enhance physical and sexual functioning in humans, by tonifying kidney yang (shen yang) and strengthening bone and tendons . The effects of deer tendons on osteoporosis and bone loss have been studied in rat models [2, 3]. Cattle tendons are commonly used as a less effective substitute. Owing to the limited supply and high demand, the price of genuine deer tendons is about five to ten times higher than that of cattle tendons . This leads to frequent substitution of deer tendons with cattle tendons by dishonest sellers. In September 2011, the Hong Kong Customs and Excise Department seized about 112 kg of suspected fake deer tendons worth about HK$41200 (US$5316) . In March 2012, the same department tested the deer tendons sampled from 29 shops, and found that the samples from 28 shops were cattle tendons .
The two types of tendons are generally differentiated by physical examination, i.e., deer tendons are usually smaller than cattle tendons and have a lighter color [5, 6]. The Hong Kong Government Laboratory developed a deer-specific PCR test to distinguish deer tendons from cattle tendons to prevent commercial fraud .
Loop-mediated isothermal amplification (LAMP) is a novel isothermal amplification technique that employs a DNA polymerase with strand-displacement activity together with a set of typically four primers recognizing six specific loci on the target DNA. The reaction and detection can be finished in one step owing to the large amounts of DNA and side products produced during the reaction. The technique has been employed to detect various pathogens in the field since its first report in 2000 . Recently, our group has applied LAMP to differentiate an herbal tea ingredient, Hedyotis diffusa Willd, from its common adulterant .
In the present study we developed a LAMP test to differentiate deer tendons from cattle tendons to facilitate on-site identification. Currently, the available drugs for treating bone loss and osteoporosis are mainly antiresorptive agents . Some drugs like bisphosphonates also decrease bone formation . Long-term use of these drugs has been associated with several side effects [10, 11]. Because both types of tendons have been used to protect bone and tendons, we evaluated the effects of extracts from the two tendon types on bone formation and bone resorption using osteoblast-like UMR106 cells and pre-osteoclast Raw264.7 cells, respectively. Both cell types are well-established models for evaluating the bone-protective effects of potential drugs [12, 13].
Reagents and chemicals
Reagents for cell culture, including medium, antibiotics, and serum, were purchased from Gibco, Thermo Fisher Scientific (Grand Island, NY, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.
DNA extraction and quantification
The tendon samples (approximately 20 mg) were cut into small pieces, and the DNA was extracted using a CTAB method . The isolated DNA was quantified by a spectrophotometer (NanoDrop, Thermo Fisher Scientific, Wilmington, DE, USA) and stored at −20 °C.
Primer design, LAMP reaction, and product detection
Information for LAMP primers
Sequence (5′ to 3′ direction)
The LAMP reaction was performed using an Isothermal MasterMix Amplification Kit (OptiGene, Horsham, UK) and set up in accordance with the manufacturer’s instructions. The mixture was incubated at 65 °C for 1 h in a PCR machine or portable Genie II LAMP Detector (OptiGene) followed by inactivation at 98 °C for 5 min. Three strategies were employed to detect the amplification: (1) real-time monitoring in the Genie II Detector using the fluorescent DNA binding dye provided in the kit; (2) post-reaction analysis by 1 % TAE gel electrophoresis; and (3) colorimetric detection under a UV lamp by staining with GelRed (Biotium, Hayward, CA, USA). The sensitivity of the LAMP test was determined by tenfold dilution of a DNA sample from deer tendon (500 ng/µL).
Preparation of tendon extracts
Tendon samples were preswollen in distilled water for 48–72 h at 4 °C, and then processed to small pieces using scissors and a blender. The tendon mixtures were digested with 1:1000 (g/g) porcine pepsin (Genview, League City, TX, USA) at 37 °C for 24 h and sonicated for 15 min, before being centrifuged at 2460×g in a J6-MI centrifuge (Beckman Coulter Brea, CA, USA) for 30 min. The supernatants were lyophilized and stored at −20 °C.
Mouse pre-osteoclast Raw264.7 cells and rat osteoblast-like UMR106 cells were obtained from ATCC (Manassas, VA, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 100 U/mL of penicillin/streptomycin at 37 °C under 5 % CO2.
Cytotoxicity and tartrate-resistant acid phosphatase (TRAP) staining in Raw264.7 cells
Raw264.7 cells were seeded in 96-well plates at 2000 cells/well and cultured overnight to allow cell attachment. The cells were further cultured with DMEM supplemented with 10 % FBS and different concentrations of tendon extracts for 4 days to determine the cytotoxicity of the tendon extracts. The medium was refreshed on day 3 (post-treatment; described below). The cytotoxicity was determined by the MTT assay as previously described . For TRAP staining, the culture medium was changed to α-MEM supplemented with 10 % FBS, 80 ng/mL of receptor activator of NF-κB ligand (RANKL) (R&D Systems, Minneapolis, MN, USA), and 100 µg/mL of tendon extract on the day after cell seeding. TRAP staining was performed on day 4 using an Acid Phosphatase Kit (Sigma-Aldrich) in accordance with the manufacturer’s instructions. Osteoclasts were determined under an inverted research IX71 microscope, (Olympus, Tokyo, Japan) by TRAP-positive staining and presence of multinucleated cells (≥3 nuclei).
Proliferation, alkaline phosphatase (ALP) activity, and calcium deposition in UMR106 cells
UMR106 cells were seeded in 96-well plates at 3,000 cells/well and cultured overnight. The culture medium was then changed to DMEM supplemented with 1 % FBS and various concentrations of the tendon extracts. DMEM containing 10 % FBS was used as a positive control. The cell proliferation was determined after 72 h of treatment using the MTT assay as described above.
UMR106 cells were seeded in 24-well plates at 10,000 cells/well and cultured overnight. The culture medium was then replaced with differentiation medium (DMEM supplemented with 10 % FBS, 50 µg/mL of ascorbic acid, and 10 mM β-glycerophosphate) containing 200–400 µg/mL of tendon extract. Cells treated with dexamethasone at 100 µM were used as a positive control. The medium was refreshed on day 3. The ALP activity was determined on day 5 using a LabAssay ALP Kit (Wako Pure Chemical Industries, Osaka, Japan) in accordance with the manufacturer’s instructions. The results were normalized by the protein contents determined using a Pierce BCA Protein Assay Kit (Pierce Biotechnology, Thermo Fisher Scientific Rockford, IL, USA).
Alizarin Red S staining was performed to detect calcium deposition. UMR106 cells were seeded in 24-well plates at 2000 cells/well and cultured overnight. The cells were treated as described for the ALP assay. On day 6, the cells were fixed with 75 % ethanol for 1 h and then stained with 40 mM Alizarin Red S for 10 min. The stain was dissolved in 10 % cetylpyridinium chloride and quantified by the absorbance at 562 nm measured in a microplate spectrophotometer (BioTek, Thermo Fisher ScientificWinooski, VT, USA).
Raw264.7 and UMR106 cells were seeded and treated as described for the TRAP staining and ALP activity evaluations, respectively. Total RNA was extracted using TRIzol reagent (Life Technologies Carlsbad, CA, USA) as previously described  and quantified by the NanoDrop spectrophotometer. Total RNA (2 µg) was reverse-transcribed into cDNA by MuMLTable S2V reverse Transcriptase (Promega, Madison, WI, USA). The primers for evaluation of the relative gene expressions are shown in Additional file 1: Table S2. Real-time PCR was performed in an ABI 7500 PCR System (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA) using a SYBR Premix Ex Taq Kit (Takara, Kusatsu, Japan) in accordance with the manufacturer’s instructions. Relative quantification was calculated by the 2−∆∆CT method.
Data were expressed as the mean ± SD of at least three independent experiments and analyzed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Differences between two groups were evaluated by Student’s t test. Values of P < 0.05 were considered statistically significant.
Results and discussion
Differentiation of deer tendons and cattle tendons by the LAMP test
Effects of tendon extracts on bone remodeling
Bone remodeling involves both osteoblast-induced bone formation and osteoclast-mediated bone resorption. The imbalance of bone remodeling leads to bone loss and osteoporosis . The effects of both types of tendons on bone remodeling in pre-osteoclast Raw264.7 cells and osteoblast-like UMR106 cells were investigated.
Relative gene expression in Raw 264.7 (A) and UMR106 cells (B)
Cattle tendon (100 µg/mL)
0.45 ± 0.17**
0.34 ± 0.11**
0.40 ± 0.18*
Deer tendon (100 µg/mL)
0.74 ± 0.14
0.55 ± 0.20*
0.72 ± 0.18
Cattle tendon (200 µg/mL)
1.28 ± 0.82
0.90 ± 0.54
1.37 ± 0.16
Cattle tendon (400 µg/mL)
1.82 ± 0.50
1.18 ± 0.27
1.58 ± 0.49
Deer tendon (200 µg/mL)
1.40 ± 0.76
0.88 ± 0.25
1.56 ± 0.66
Deer tendon (400 µg/mL)
1.72 ± 0.86
0.82 ± 0.20
2.09 ± 0.96*
The dried mass of tendons consists of 65–80 % collagen (mostly type I collagen) and less than 35 % non-collagenous proteins . Type I collagen is highly conserved in land mammals . The similarity in protein compositions limits the application of protein-based analysis for differentiation between the tendon types. In the present study, the LAMP test could differentiate deer tendons from cattle tendons. Compared with the specific PCR test developed by the Hong Kong Government Laboratory , we have introduced an internal control reaction to reduce false-negative results in the specific reaction. Generally, DNA is more or less degraded in dried products, and this leads to negative amplification even in authenticated samples . An internal control reaction to detect both types of tendons can avoid such false-negative results. Besides the conventional gel electrophoresis detection, we also introduced real-time observation and colorimetric detection, which will facilitate on-site detection. Above all, the LAMP reaction is performed at a constant temperature and can be carried out in water kept in a thermal bottle . The isothermal DNA amplification test combined with the fast DNA extraction procedure is easy to adopt by retailers and consumers.
In the present study, both deer tendons and cattle tendons exhibited similar effects in inhibiting the differentiation of Raw264.7 cells into osteoclasts and promoting the cell proliferation and mineralization of UMR106 cells. The collagens from deer tendon and deer antler are effective against osteoporosis in ovariectomized rats [2, 3, 29]. To our knowledge, there have been no reports characterizing the biological effects of cattle tendons either in vivo and in vitro. The results of the present study could not determine whether the beneficial effects of the tendon extracts were caused by the collagenous or non-collagenous fractions. Unlike herbal medicines that have standard protocols for their partition and isolation, crude tendon extracts are mixtures of many proteins and other molecules. We have started to characterize the effective components in the tendon extracts and to compare the beneficial effects of the two types of tendon extracts in ovariectomized rat models.
This study demonstrated that the developed isothermal LAMP test can distinguish between deer tendons and cattle tendons. Biological assays showed that both types of tendons exhibited similar beneficial effects on bone remodeling.
PCS conceived the study. LLJ, CLL and PCS designed the study. LLJ, CLL performed the cell assays. YLW and CFN performed the LAMP tests. LLJ and PCS wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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