CN112795671B - Nested PCR (polymerase chain reaction) primer, kit and detection method for detecting enterococcus faecium - Google Patents
Nested PCR (polymerase chain reaction) primer, kit and detection method for detecting enterococcus faecium Download PDFInfo
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Abstract
The invention discloses a nested PCR primer, a kit and a detection method for detecting enterococcus faecium. The invention designs a primer to carry out nested PCR on a sample to be detected according to an RNA polymerase beta subunit which is conserved in enterococcus and is dependent on single copy gene DNA and found by bioinformatics analysis. The method avoids the influence on the PCR detection rate due to variation of enterococcus faecium strains of the tussah silkworm, can quickly monitor silkworm diseases and carry out early preventive treatment due to the high sensitivity and specificity of nested PCR, can even detect egg surfaces, silkworm tools and silkworm rooms through testers, can monitor external silkworm eggs and detect the disinfection effect of the silkworm tools and the silkworm rooms, provides an effective prevention and control means for avoiding infection of external sources or cross infection of internal sources of the tussah silkworm streptococcus, and can even become an important component of the standardized breeding process of the silkworm industry.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nested PCR primer, a kit and a detection method for detecting enterococcus faecium.
Background
Antheraea pernyi (Antheraea pernyi) ((Enterococcus pernyi) Is a pathogen causing tussah empty carcass disease and castor silkworm soft rot, and is transmitted through excreta of polluted eggs, polluted silkworm tools, wild silkworm moths with bacteria, silkworm excrement and the like. The newly hatched silkworm takes the enterococcus pernyi down by eating or contacting with the egg shell with bacteriaInfected or healthy silkworms contacting with the silkworm excrement with bacteria, thin excrement and vomit liquid contain a large amount of enterococcus fassae, and pollute feeds, silkworm utensils and silkworm rearing rooms for cross infection. Moreover, enterococcus faecium can be transmitted by flies, and researches show that the enterococcus faecium has strong resistance to ultraviolet rays, so that the infection control becomes very troublesome.
In the whole growth and development process of tussah, the tussah can be damaged by enterococcus faecium from 1 to 5 years old, and has symptoms, particularly 1, 2 and 3 sleeps and the onset of dormancy is more. After the tussah is sleeped, the tussah is eaten as usual, the disease starts to occur after 2-3 days, the main manifestations are slow in movement, the tussah stays on the tussah branches, the tussah does not like to eat leaves, the growth is slow, the silkworm body is thin and slightly contracted, and the digestion promoting tube is hollow and is free of hard excrement granules. Dark brown thin feces is often excreted, and the anus is polluted by the thin feces. The disease condition is further developed, the silkworm body is bigger in head and smaller in tail, the setae are long, and finally the diseased silkworm holds the oak branches with the tail feet or the tail feet and the ventral feet, and the head and the chest of the diseased silkworm droop and die. The dead corpse is not festered and has no fishy smell and bad smell.
The castor silkworm can be infected by enterococcus faecium in the whole instar period, and the infection symptoms of the larvae in different instar periods are different. The contamination of silkworm tool or egg surface can make the Antheraea pernyi enterococcus infect whole batch of castor silkworm, the disease does not occur in 1 year, the disease occurs in 2 years after molting, the leaf eating is not favored, the silkworm body is thin and weak, the excrement is discharged, the vomiting is caused, finally the silkworm body collapses and dies, and the dead silkworm body becomes black. Infection symptoms of the old silkworms can not be killed, the silkworm bodies are excreted and excrement is accompanied by odor, pupae can be transformed but are generally malformed, the pupae can be directly transformed without spinning, the eclosion rate of the malformed pupae is reduced and is often accompanied by the phenomenon of failure of the eclosion, and destructive impact is brought to production.
The infection of the enterococcus faecium of tussah is mainly prevented and assisted by treatment, once the silkworm is attacked, the silkworm is difficult to cure, the production of the next generation is influenced, and the cross contamination of the next generation can be caused. However, no effective monitoring method for enterococcus faecium has been reported.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a nested PCR primer, a kit and a detection method for detecting enterococcus faecium.
The first purpose of the invention is to provide a nested PCR primer for detecting enterococcus faecium, which comprises the following primers:
outer primer:
EperDDRP β F: 5'-CTCAAGGTCTTCCTCGTGTCCA-3' (shown in SEQ ID NO. 2), EperDDRP beta R: 5'-ACTTTACGAAGCATTTGGCGAAC-3' (shown in SEQ ID NO. 3);
inner primer:
EperDDRP beta U: 5'-ATCTTTGAAGCACGTAATCCGAA-3' (shown in SEQ ID NO. 4), EperDDRP beta L: 5'-GCCGATTTCTACCCCTTGCAT-3' (shown in SEQ ID NO. 5).
The second purpose of the invention is to provide a kit for detecting enterococcus faecium, which comprises the nested PCR primer.
The third purpose of the invention is to provide a method for detecting the enterococcus faecium, which comprises the following steps: extracting DNA of a sample to be detected as a template, performing first round PCR amplification by using the outer primers EperDDRP beta F and EperDDRP beta R, performing second round PCR amplification by using the inner primers EperDDRP beta U and EperDDRP beta L and detecting a first round PCR amplification product and a second round PCR amplification product by using agarose gel electrophoresis, wherein if only the second round PCR amplification product has 1 electrophoresis strip, or if both the first round PCR amplification product and the second round PCR amplification product have 1 electrophoresis strip, the sample to be detected contains enterococcus faecium pernyi.
Preferably, the PCR system of the first round of PCR amplification is 50 μ L, and comprises 1 μ L of Taq enzyme, 2.5 μ L of 2.5 mM dNTPs, 5 μ L of 10 XBuffer, 1 μ L of 10 μ M EperDDRP β F, 1 μ L of 10 μ M EperDDRP β R, 10 μ L of DNA template, and the balance of sterile deionized water.
Preferably, the PCR system of the second round of PCR amplification is 50 μ L, and comprises 1 μ L of Taq enzyme, 2.5 μ L of 2.5 mM dNTPs, 5 μ L of 10 XBuffer, 1 μ L of 10 μ M EperDDRP β U, 1 μ L of 10 μ M EperDDRP β L, 1 μ L of the first round PCR amplification product, and the balance of sterile deionized water.
The PCR amplification conditions of the first round of PCR amplification and the second round of PCR amplification are both 94 ℃ pre-denaturation for 2 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; extension at 72 ℃ for 7 min.
The fourth purpose of the invention is to provide the application of the nested PCR primer for detecting the enterococcus faecium or the kit for detecting the enterococcus faecium in the detection of the enterococcus faecium.
The invention designs a primer to carry out nested PCR on a sample to be detected according to an RNA polymerase beta subunit which is conserved in enterococcus and is dependent on single copy gene DNA and found by bioinformatics analysis. The method avoids the influence on the PCR detection rate due to variation of enterococcus faecium strains of the tussah silkworm, can quickly monitor silkworm diseases and carry out early preventive treatment due to the high sensitivity and specificity of nested PCR, can even detect egg surfaces, silkworm tools and silkworm rooms through swabs, monitor external silkworm eggs and detect the disinfection effect of the silkworm tools and the silkworm rooms, provides an effective prevention and control means for avoiding infection of external sources or cross infection of internal sources of the tussah silkworm streptococcus, and even can become an important component of the standardized breeding process of the silkworm industry.
The nested PCR primer has higher sensitivity and specificity, and is used for accurately detecting infection, pollution, residue and the like of enterococcus fassae in environments such as diseased silkworms, silkworm eggs, silkworm excrements, silkworm rearing instruments, silkworm rearing rooms and the like of giant silkworms such as tussah silkworms and castor silkworms. The method has the advantages of high detection speed and capability of sensitively monitoring the enterococcus faecium of the tussah so as to realize accurate diagnosis and prevention of the empty carcass disease of the tussah and the soft rot disease of the castor silkworm.
Drawings
FIG. 1 is a flow chart of the detection of enterococcus faecium according to the present invention.
FIG. 2 shows the results of the electrophoretogram of example 2.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
gene sequences were predicted by genome-wide annotation of 51 bacterial genomes of enterococcus, wherein the sequence number of enterococcus pernyi at NCBI is NZ-KV 388085.1. Then 51 genes of the bacterial genome were clustered to predict orthologous genes, and single-copy orthologous genes were selected. By comparing 462 sets of single copy homologous genes, the longest gene is DNA-dependent RNA polymerase beta subunit (DNA-directed RNA polymerase subunit beta) (sequence name: NZ _ KV388085.1:2538177-2541830 (+); sequence source: NCBI sequence number NZ _ KV388085.1, base 2538177 to base 2541830 of the positive strand, as shown in SEQ ID NO. 1), and the template is designed according to the sequence as a primer. The annealing temperature of the primer is designed to be about 60 ℃, and the length of the amplified product is within 400 bp, so that the method is suitable for fluorescent quantitative PCR. Wherein EperDDRP beta F and EperDDRP beta R are outer primers, EperDDRP beta U and EperDDRP beta L are inner primers, and specific primer sequences are shown in Table 1. TABLE 1 primer sequence Listing
The 4 primers can be obtained by a conventional synthesis method.
The flow chart of the implementation of the nested PCR primer for detecting the enterococcus faecium is shown in figure 1.
The method comprises the following specific steps:
1. collecting samples: the collected sample comprises silkworm tissue, silkworm egg, silkworm secretion, silkworm tool, silkworm chamber swab and the like. The swab can be used for wiping silkworm tool, silkworm room, silkworm egg surface, silkworm body surface, silkworm secretion, etc. with cotton swab, absorbent cotton ball, paper towel ball, etc.
2. DNA extraction: the DNA extraction method can be CTAB method, SDS method or protease method.
3. First round PCR: the PCR primers are outer primer pairs EperDDRP beta F (shown as SEQ ID NO. 2) and EperDDRP beta R (shown as SEQ ID NO. 3), the final concentration of the primers is 0.2 mu M, and the first round of PCR amplification is carried out on the DNA of a sample to be detected. The amplification condition is pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 25-35 times; extension at 72 ℃ for 7 min.
4. Second round PCR: the PCR primers are inner primer pairs EperDDRP beta U (shown as SEQ ID NO. 4) and EperDDRP beta L (shown as SEQ ID NO. 5), the final concentration of the primers is 0.2 mu M, the first round of PCR amplification products can be diluted by 50-200 times as required, and the primers can also be directly used as templates for second round PCR amplification. The amplification condition is pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 25-35 times; extension at 72 ℃ for 7 min.
5. And (3) detection results: the first round of PCR and the second round of PCR can detect the amplification result by a real-time fluorescent quantitative PCR method and can detect the amplification result by 1.5 percent agarose gel electrophoresis.
Example 2: detecting silkworm room with 3 months outbreak of Antheraea pernyi in Nanjing
1. Collecting swabs: wiping different positions of the silkworm chamber 5 and wiping excrement of silkworms at the position 3 with a paper towel ball to prepare swabs.
2. DNA extraction: a50 mL centrifuge tube was placed in a swab, and the swab was soaked in 5 mL of 2 XTAB extraction buffer (composition: 2% (w/v) CTAB, 100 mM Tris (pH 8.0), 20 mM EDTA, 1.4M NaCl, 0.2% (v/v). beta. -mercaptoethanol) and water-washed at 65 ℃ for 2 hours. After cooling for 2 minutes, 5 mL of a phenol-chloroform-isoamyl alcohol mixture (25: 24: 1) was added, and the mixture was vigorously shaken and mixed. Centrifuge at 12000 rpm for 5 minutes. Taking the supernatant into a new centrifuge tube, adding isopropanol with the same volume, and reversing and mixing uniformly. The mixture was precipitated at-20 ℃ for 30 minutes or more, centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was washed with 75% ethanol 1 time, centrifuged at 12000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was naturally dried and dissolved in 50. mu.L of sterile deionized water.
3. First round PCR: mu.L Taq enzyme, 2.5. mu.L 2.5 mM dNTPs and 5. mu.L 10 are added into the PCR systemEasyTaq ®Buffer, 1. mu.L 10. mu.M EperDDRP. beta.F, 1. mu.L 10. mu.M EperDDRP. beta.R, 10. mu.L DNA template, sterile deionized water to 50. mu.L. The PCR amplification condition is pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; extension at 72 ℃ for 7 min.
4. Second round PCR: mu.L Taq enzyme, 2.5. mu.L 2.5 mM dNTPs and 5. mu.L 10 are added into the PCR systemEasyTaq ®Buffer, 1 uL 10 uM EperDDRP beta U, 1 uL 10 uM EperDDRP beta L, 1 uL first round PCR amplification product, and sterile deionized water is added to 50 uL. The PCR amplification condition is pre-denaturation at 94 ℃ for 2 min; the denaturation is carried out for 30s at the temperature of 94 ℃,annealing at 60 ℃ for 30s, extending at 72 ℃ for 30s, and circulating for 35 times; extension at 72 ℃ for 7 min.
5. And (3) detection results: configuring 1.5% agarose gel, electrophoresis at 125V voltage for 30 minutes, and observing bands of the first round PCR and the second round PCR amplification products. As a result, as shown in FIG. 2, the numbers before the electrophoretic well marked "-" indicate swab numbers, the number after "-" 1 indicates a first round PCR amplification product, the number after "-" 2 indicates a second round PCR amplification product, the swabs No.1 to 5 indicate chamber swabs, and the swabs No. 6 to 8 indicate silkworm excrement swabs. The result shows that the No. 1-5 silkworm chamber swabs can detect the pollution of the enterococcus faecium, the first round PCR amplification products do not have electrophoresis bands, the second round PCR amplification products can clearly detect 3 target bands, and the No.2 and No.5 silkworm chamber swabs do not detect the enterococcus faecium. The first round PCR amplification products of No. 6-8 silkworm excrement swabs have clear bands, which indicates that excrement contains a large amount of enterococcus fassae.
Sequence listing
<110> institute of animal research of academy of sciences of Guangdong province
<120> nested PCR primer, kit and detection method for detecting enterococcus faecium
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ttgatcgatg taaataaatt cgaaagtatg caaataggtt tggcttctcc cgaaaaaatc 60
agaagctggt cttacggtga agtaaaaaaa cctgagacga ttaactatcg taccttaaaa 120
cctgagcgcg aagggttgtt ctgtgagcgg atttttggtc ctacaaaaga ctgggaatgt 180
gcctgcggaa aatacaaacg tatccgttat aaaggaatcg tttgtgatcg ctgtggtgtg 240
gaagtgactc gttcaaaagt tcgtcgtgaa cgtatgggtc atatcgaatt agcagcacct 300
gtttcacata tctggtattt caaaggtatt ccttctcgta tgggtcttgt attagacatg 360
agcccacgcg cattagaaga aatcatttac tttgcctcat atgttgttat tgaaccagga 420
aacacaagct tagagaaaaa acaattgttg actgaacgcg aataccgcga aaaacgtgaa 480
caatatggcc aagaattcca agcagcaatg ggcgcggaag caatcaaaca attacttgat 540
aacgttgatt tggatggcga agcggctgaa ttgaaagaag agttgaaatc tgcacaagga 600
caaaaacgta cacgcgcgat ccgtcgtttg gacattttag aagcattccg tgcttctggc 660
aatgaaccaa gctggatggt catggacgtt atccctgtca tcccaccaga tctacgacca 720
atggtccaat tagaaggtgg acgttttgca acaagtgact tgaacgatct atatcgtcgt 780
gtgatcaacc gtaacaatcg tttgaaacgt ttgttagact tgaatgcacc aggaatcatc 840
gttcaaaatg aaaaacggat gttacaagaa gcggtagatg cgttgatcga taacggtcgt 900
cgtggccgtc ctgttacagg accaggtaac cgcccattga aatctctttc tcatatgttg 960
aaagggaaac aaggtcgttt ccgtcaaaac ttacttggta aacgtgtcga ctactctggt 1020
cgttcagtta tcgtcgtagg tcctttcttg aaaatgtacc aatgtggatt accaaaagaa 1080
atggcgatcg aattgttcaa accattcgtg atgcgcgaat tagttcaacg tgaacttgct 1140
tcaaacatca aaaatgcgaa acgcaaaatc gaacgtcaag aagatgaagt ttgggatgtc 1200
ttagaagacg tcatcaaaga acatccagtt ctattgaacc gggcacctac gcttcacaga 1260
ttaggtatcc aagcatttga accagttctt gtccaaggtc gtgcgatccg tttgcatcca 1320
ttggtatgtg aagcttataa tgccgatttc gatggagacc aaatggccgt tcacgtaccg 1380
ttgaacgaag aagcacaagc agaagcacgt atgctgatgt tagctgctca aaacatcttg 1440
aatccaaaag atggtaaacc agttgttaca ccttctcagg atatggtttt aggaaactac 1500
tacctaacaa tggaagaaga aggtcgtgaa ggcgaaggaa tggtctttag ggaccaaaac 1560
gaagcagtca tcgcatggcg taatggctac gttcatttac attcacgtat tggtgtgaac 1620
ccaacgacat taggggacaa accatttaca gaatggcaaa aagaacgcac gatgatcacg 1680
actgttggta agatcatctt taacgagatc atgccaccag aattccctta cttgaatgag 1740
ccaactgact tcaacttgac tgtccaaaca ccggacaaat actttgttga agctggaaca 1800
gatattcctg ctcacatcaa agaacaagaa ctagttttac cattcaagaa gaaaaactta 1860
ggaaacatca tcgccgaagt cttcaaacgt ttcaaagtaa ctgaaacatc taagatgctt 1920
gaccgtatga aagacttagg atacaaacat tcaacacatg caggtatcac tgtagggatc 1980
gccgacatca gtgtcttgaa tgaaaaacaa gtgatcattg acgaagcaca taaacaagta 2040
gagtcgatca cgaagcaatt ccgtcgtgga ttgatcactg acgacgagcg ttatgaacga 2100
gtaatcgctg tttggaactc tgcgaaagac agcatccaac aaaaactgat ggaaggtttg 2160
gaagcaaaaa acccaatctt catgatgtca gattctggtg cccgtggtaa catctccaac 2220
tttactcagc ttgctggtat gcgtggattg atggccgcac cgaatggacg tatcatggaa 2280
ttgccgatca tttcgaactt ccgcgaagga ctttctgtct tggaaatgtt tatctcaact 2340
cacggggctc gtaaaggaat gaccgataca gccttgaaga cagccgattc tggttacttg 2400
acacgtcgtt tagttgacgt tgcccaagat gtcatcattc gtgaagaaga ttgtggcact 2460
gaccgcggtc ttgagatcca agcaatccgt gaaggaaatg aaatcatcga atctcttgaa 2520
gatcgtttga ttggtcgtta cacacgtaaa gaagttcgtc atcctgaaac caacgaaatg 2580
atcatcgccg gaaatcaatt gatctctgaa gatgttgcaa aacaaatcgt tgatgcaggt 2640
gttgaaactg taacgatccg ttctgtcttc acatgtaaca caaaacatgg tgtctgcaaa 2700
cattgttacg gacgtaactt ggcaactggt tctgacgttg aagtcggaga agcagttggt 2760
acgatcgccg ctcaatcaat cggtgaacca ggtactcagt taacaatgcg tacgttccat 2820
acgggtgggg ttgccggaga tgatatcact caaggtcttc ctcgtgtcca agaaatcttt 2880
gaagcacgta atccgaaagg gcaagcagtg attactgaag taacagggga tgtcatcgat 2940
atctctgaag atcctgctac acgtaccaaa gaagtaacaa tcaaagggaa aacagataca 3000
cgtacgtata cagtgcctta tactgcacgt atgaaagtcg ctgaaggcga tatgatccat 3060
cgtggtgcac cattgacaga aggatcaatc gatccgaaac aattgttgca agtacgtgac 3120
gttctatctg ttgaaaacta tctattacgt gaagtacaac gcgtttaccg tatgcaaggg 3180
gtagaaatcg gcgacaaaca tatcgaagta atggttcgcc aaatgcttcg taaagtgcgc 3240
gtcatggacc caggcgatac agaaatcttg ccaggtacac tgttagatat cgcagacttc 3300
aaagaccaaa actacaatac gttagttgct ggtggcgttc ctgcgacatc tcgtccagtc 3360
ttgctaggta tcacaaaagc atcattagaa acaaacagct tcttatcagc tgcatccttc 3420
caagaaacaa ctcgtgtctt gactgatgct gcgatccgag gcaaaaaaga tccacttctt 3480
ggtttgaaag aaaatgttat catcggtaag atcattccag ctggtactgg tatgccacgt 3540
taccgcaaca tggaaccaaa agaagttggc gttgcaagtg aaaacgtgta tagtatttct 3600
gacatcgaag cacaaatggc tgccgaagat gcaatgaatg aacaaacacg ataa 3654
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<213> Artificial Sequence (Artificial Sequence)
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ctcaaggtct tcctcgtgtc ca 22
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<213> Artificial Sequence (Artificial Sequence)
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actttacgaa gcatttggcg aac 23
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<213> Artificial Sequence (Artificial Sequence)
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atctttgaag cacgtaatcc gaa 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gccgatttct accccttgca t 21
Claims (7)
1. A nested PCR primer for detecting enterococcus faecium is characterized in that the nucleotide sequence of the nested PCR primer is as follows:
outer primer:
EperDDRPβF：5’-CTCAAGGTCTTCCTCGTGTCCA-3’，EperDDRPβR：5’-ACTTTACGAAGCATTTGGCGAAC-3’；
inner primer:
EperDDRPβU：5’-ATCTTTGAAGCACGTAATCCGAA-3’，EperDDRPβL：5’-GCCGATTTCTACCCCTTGCAT-3’。
2. a kit for detecting enterococcus faecium, which comprises the nested PCR primer of claim 1.
3. A method for detecting enterococcus faecium for non-disease diagnosis is characterized by comprising the following steps: extracting DNA of a sample to be detected as a template, performing first round PCR amplification by using the outer primers EperDDRP beta F and EperDDRP beta R as claimed in claim 1, performing second round PCR amplification by using the inner primers EperDDRP beta U and EperDDRP beta L as claimed in claim 1 and using agarose gel electrophoresis to detect the first round PCR amplification product and the second round PCR amplification product, wherein if only the second round PCR amplification product has 1 electrophoresis band, or if both the first round PCR amplification product and the second round PCR amplification product have 1 electrophoresis band, the sample to be detected contains enterococcus faecium.
4. The method for detecting enterococcus faecium according to claim 3, wherein the PCR system of said first round of PCR amplification is 50 μ L, comprising 1 μ L Taq enzyme, 2.5 μ L2.5 mM dNTPs, 5 μ L10 XBuffer, 1 μ L10 μ M EperDDRP β F, 1 μ L10 μ M EperDDRP β R, 10 μ L DNA template, and the rest is sterile deionized water.
5. The method for detecting enterococcus faecium according to claim 3, wherein the second round of PCR amplification has a PCR system of 50 μ L, and comprises 1 μ L of Taq enzyme, 2.5 μ L of 2.5 mM dNTPs, 5 μ L of 10 XBuffer, 1 μ L of 10 μ M EperDDRP β U, 1 μ L of 10 μ M EperDDRP β L, 1 μ L of the first round PCR amplification product, and the balance of sterile deionized water.
6. The method for detecting enterococcus faecium according to claim 3, 4 or 5, wherein the PCR amplification conditions of the first PCR amplification and the second PCR amplification are pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; extension at 72 ℃ for 7 min.
7. The use of the nested PCR primer for the detection of enterococcus faecium of tussah according to claim 1 or the kit for the detection of enterococcus faecium of tussah according to claim 2 for the detection of enterococcus faecium of tussah for non-disease diagnosis purposes.
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