CN101469341B - Identification plate for gram negative aerobic bacteria and preparation thereof - Google Patents

Abstract

The invention provides an identification plate for Gram-negative aerobic bacteria. The combination of 47 carbon sources and nitrogen sources on the identification plate can identify more than 300 kinds of Gram-negative bacteria, can identify various bacteria such as negative Enterobacteriaceae, Vibrionaceae, pseudomonas, Acinetobacter, stenotrophomonas, negative fastidious bacteria, and so on, can obtain an identification result without a supplementary experiment, and has simple operation and accurate results, so the identification plate has higher clinical application value. The invention provides a method for preparing the identification plate for the Gram-negative aerobic bacteria.

Description

Identification plate for gram negative aerobic bacteria and preparation method thereof

Technical field:

The present invention relates to biotechnological formulation.Be specifically related to a kind of identification plate for gram negative aerobic bacteria and preparation method thereof.

Background technology

Over nearly 20 years; Owing to extensively use miscellaneous antibacterials in large quantities clinically, drug-resistance of bacteria is more and more general, and the bacterium that has is anti-2-7 kind medicine simultaneously; The wide-scale distribution of Resistant strain is brought very big difficulty to clinical treatment, particularly for the bacterium of nosocomial infection.So the infection of the characteristics of nosocomial infection in appearing at hospital environment, and take place popular, so can be referred to as hospital acquired infections (Hospital acquired infection) again.Though the state of an illness of generation hospital infection has and gently has heavily, has all increased additional pain, economical load and misfortune to patient, also can increase the weight of the burden of medical treatment and nursery work simultaneously owing to the prolongation hospital stays, influence sick bed and have enough to meet the need.The disease that serious hospital infection often makes patient is suffered from can not reach the curative effect of expection, even produces sequela or the death that is difficult to treat.According to an incomplete statistics data report of the U.S., have 5% hospital infection takes place among its inpatient approximately, the average 4～5d that is in hospital that prolongs has 100,000 people's death every year approximately in hospital infection, estimate annual since hospital infection to consume 20 more surplus hundred million dollars.

From causing the mikrobe of hospital infection; No doubt some is still the pathogenic very strong mikrobe that we are familiar with; But the pathogenic agent kind of hospital infection in recent years has the trend of considerable change; It was the gram-positive cocci of main pathogen originally that Gram-negative bacteria has replaced, and wherein enterobacteriaceae and pseudomonas account for the 60%-65% of hospital infection, and was mainly many drug tolerant bacterias.Owing to use microbiotic widely, the continuous transition of bacterium have encouraged the existence and the development of nosocomial infection, and the clinical microbiology most important character of today is to change to the hospital infection aspect.

Microorganism identification and susceptibility analytical system are the high-tech in-vitro diagnosis products that is mainly used in clinical labororatory, and be significant to auxiliary clinical each section office's diagnoses and treatment.At present in the world, Rapid identification and the susceptibility analysis of bacterium obtained very big progress, mikrobe robotization testing product has progressively substituted manual inspection, and domestic similar research still is in the exploratory stage.

At present, like product mainly contains in the world: the MicroScan automatic microbe of the VITEK-AMS microbiological analysis system of French biology-Mei Liai company, U.S. Dade-MicroScan company is identified and the SENSTITRE fluorescent method fast microbiological of antibiotics susceptibility test system and Britain Trek Diagnostic System Ltd. is identified and the susceptibility analytical system.Their principle of work is close, mainly is the difference according to the bacterium physico-chemical property, adopts photoelectric colorimetry, measures bacterial growth and decomposes substrate and cause pH value to change and the different colours that produces and turbidity change and judge response situation, thereby provide the identification and susceptibility result.

Domestic also have minority producer researching and developing analogous products, like microflora of the world, Hunan people etc.But because this intermediate item technology content is higher, fund input is big, and especially the accumulation in bacterium storehouse takes time and effort, so present domestic analogous products are also very immature.On clinical expansion, mainly there is the problem of the following aspects in microorganism identification:

(1) qualification time is long partially, needs to provide in 16-24 hour the result;

(2) evaluation scope is narrow, kind surplus the bacterium that can identify has only 300.

Summary of the invention

Technical problem to be solved by this invention is to overcome above-mentioned weak point, a kind of identification plate for gram negative aerobic bacteria quick and precisely of research and design.

Identification and detection of the present invention is tested based on following principle:

According to present microorganism identification field authority's the outstanding Bacteria Identification handbook of uncle, the evaluation of bacterium mainly through the utilize situation of bacterial detection to various nutritive substances, is promptly identified bacterium through bacterial detection biochemical reaction characteristic.And bacterium utilizes in the process of nutritive substance generation biochemical reaction; A series of redox reaction can take place; Can produce NADH in these redox reactions, and NADH can be tetrazolium violet (2,3; 5-triphenyl tetrazolium chloride, TTC) indicator is changed into the reduced form of purple by colourless oxidized form.Therefore we use the indicator of tetrazolium violet as bacterium generation biochemical reaction, come the biochemical reaction characteristic of bacterial detection, thereby reach the purpose of Bacteria Identification.

Different bacterium utilizes different carbon sources or nitrogenous source to get into metabolic processes, then can't utilize some other carbon source or nitrogenous source, and these characteristics are that the genetic material of bacterium determines.Present existing microorganism identification plate has only 30 biochemical reactions at most; Therefore narrow for the kind aspect of identifying bacterium; Kind surplus the gram-negative bacteria most species that can identify has only 200; And different types of bacterium need use different types of lath to identify, need can draw qualification result through additional the checking just simultaneously, operates more loaded down with trivial details.

In order to overcome above-mentioned weak point, reach and identify that we require the purpose of bacterium, need to select the carbon source and the nitrogenous source of an appropriate on the one hand.Therefore the inventor has selected 500 several kinds of carbon source and nitrogenous source, and various bacteria is detected, and through the analysis and arrangement to these data, finally confirms the carbon source of present lath and the combination (seeing the following form) of nitrogenous source.47 carbon sources on this kind identification plate and nitrogenous source make up appraisable gram-negative bacteria kind have 300 surplus kind; Can identify multiple different types of bacteriums such as negative enterobacteriaceae, vibrionaceae, Rhodopseudomonas, acinetobacter, narrow food zygosaccharomyces, negative severe bacteria; And need not replenish experiment and just can draw qualification result, simple to operate.

Identification plate for gram negative aerobic bacteria substrate table

The invention provides a kind of identification plate for gram negative aerobic bacteria, this identification plate is made up of reaction substrate stoste and broth culture, and position and the concentration of each composition thereof of said reaction substrate stoste on identification plate is: (g/ml)

The position	The solution title	Concentration
			?A1，A7	Water	100％
?A2，A8	Tween 80	6％
			?A3，A9	N-ethanoyl-D-galactosamine	2％
?A4，A10	The L-pectinose	4％
			?A5，A11	The D-arabitol	4％
?A6，A12	The D-cellobiose	4％

?B1，B7	D-fructose	8％
			?B2，B8	The D-semi-lactosi	4％
?B3，B9	Alpha-D-glucose	10％
			?B4，B10	Glycerine	2％
?B5，B11	α-D-lactose	4％
			?B6，B12	SANMALT-S	4％

?C1，C7	D-N.F,USP MANNITOL	?8％
			?C2，C8	The D-melibiose	?4％
?C3，C9	Beta-methyl-D-glycoside	?4％
			?C4，C10	The D-psicose	?4％
?C5，C11	The D-raffinose	?4％
			?C6，C12	The D-Sorbitol Powder	?4％

?D1，D7	Sucrose	4％
			?D2，D8	Turanose	4％
?D3，D9	Pyruvic acid methyl fat	4％
			?D4，D10	The monomethyl succsinic acid	4％
?D5，D11	The cis equisetic acid	2％
			?D6，D12	Two hydration trisodium citrates	2％

?E1，E7	Sodium formate	2％
			?E2，E8	2%DL-Alpha-hydroxy Sodium propanecarboxylate	2％
?E3，E9	DL-beta-hydroxy-butanoic acid sodium	2％
			?E4，E10	Somsanit	2％
?E5，E11	The 2%P-hydroxyl phenylacetic acid	2％
			?E6，E12	D, the L-Sodium.alpha.-hydroxypropionate	2％

?F1，F7	Propanedioic acid	?2％
			?F2，F8	The acid of D-granulated sugar	?2％
?F3，F9	The amino hydrochloride of L-L-Ala	?4％
			?F4，F10	The L-L-Ala	?2％
?F5，F11	The L-alanyl-glycine	?2％
			?F6，F12	Altheine acid	?4％

?G1，G7	The L-Sodium Glutamate	4％
			?G2，G8	The L-L-Histidine hydrochloride	2％
?G3，G9	Hydroxyl-L-proline(Pro)	2％
			?G4，G10	The L-leucine	3％
?G5，G11	The L-Pyrrolidonecarboxylic acid	2％
			?G6，G12	The D-Serine	2％

?H1，H7	γ-An Jidingsuan	?2％
			?H2，H8	Inosine	?2％
?H3，H9	Thymine deoxyriboside	?2％
			?H4，H10	The putrescine dihydrochloride	?2％
?H5，H11	D, L-α-Phosphoric acid glycerol esters disodium	?2％
			?H6，H12	Alpha-D-glucose-1-phosphoric acid salt	?2％

Said broth culture is made up of following ingredients: (v/v)

The material title	Proportion
		Pure water	65.8％
GN/MT	22.4％
		The GN mixed solution	2.24％
0.5mM the folic acid aqueous solution	0.89％
		The aqueous solution of protease of 10%-12%	1.49％
The 5% yeast extract paste aqueous solution	0.1％
		The Viola crystallina aqueous solution of 0.8%-1.1%	7.17％

Wherein GN/MT (gram-negative bacteria salt storage liquid) is made up of following ingredients: (weight % concentration)

The material title	Proportion
		Pure water [H 2O]	61.082％
Two hypophosphite monohydrate sodium dihydrogen [NaH 2PO 4-2H 2O]	10.6％
		Sodium phosphate, dibasic [Na 2HPO 4]	18.7％
Repone K [KCl]	7.5％
		Ammonium sulfate [(NH 4) 2SO 4]	2.1％
Sodium pyrophosphate decahydrate [Na 4P 2O 7-10H 2O]	0.018％

Another object of the present invention has provided the preparation method of identification plate for gram negative aerobic bacteria.

Because identification plate for gram negative aerobic bacteria can be identified different types of bacterium, comprises some nutrient ingredient requirements than higher severe bacteria, therefore must there be a kind of suitable nutritive substance to satisfy the needs of various different sorts bacterial growths.

The inventor is grouped into the one-tenth in the mix reagent through following experiment and selects, and has developed a kind of mix reagent, and this mix reagent is mixed by the required nutritive substance of various bacteria growth and forms.

1, experimental technique

Choose various bacteria growing nutrient materials such as inorganic salt basis, amino acid, phosphoric acid buffer, VITAMINs, peptone, Carnis Bovis seu Bubali cream, malt extract and be mixedly configured into reagent by different combinations mode and proper proportion; Choose different types of bacterium then and make an experiment, to confirm to close the growth that kind of array mode can satisfy various different sorts bacteriums.

2, experimental result

This experiment chosen escherichia coli, enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, wood sugar oxidation Alcaligenes,, Neisseria meningitidis, Vibrio parahaemolyticus, influenzae different sorts bacterium add in the evaluation lath of different mix reagents configurations and experimentize, experimental result is following:

Show through above-mentioned experiment: the mix reagent of combination 5 possibly satisfy the needs of different sorts bacterial growth; Therefore the inventor has confirmed the array mode of mix reagent, and each material of this mix reagent is configured to GN (gram-negative bacteria) mix reagent by following mixed: weight % concentration

The material title	Proportion
		Pure water [H 2O]	75％
Inorganic salt	6.5％
		Amino acid	8％
Y factor	7.5％
		Carnis Bovis seu Bubali cream	0.5％
Phosphoric acid buffer	1％
		Peptone	1.5％

The inventive method comprises the following steps:

1, configuration identification plate reaction substrate stoste

Require the stoste of each reaction substrate of configuration according to following configuration concentration: (g/ml)

The position	The solution title	Concentration
			?A1，A7	Water	100％
?A2，A8	Tween 80	6％
			?A3，A9	N-ethanoyl-D-galactosamine	2％
?A4，A10	The L-pectinose	4％
			?A5，A11	The D-arabitol	4％
?A6，A12	The D-cellobiose	4％

?B1，B7	D-fructose	8％
			?B2，B8	The D-semi-lactosi	4％
?B3，B9	Alpha-D-glucose	10％
			?B4，B10	Glycerine	2％
?B5，B11	α-D-lactose	4％
			?B6，B12	SANMALT-S	4％

?C1，C7	D-N.F,USP MANNITOL	?8％
			?C2，C8	The D-melibiose	?4％
?C3，C9	Beta-methyl-D-glycoside	?4％
			?C4，C10	The D-psicose	?4％
?C5，C11	The D-raffinose	?4％
			?C6，C12	The D-Sorbitol Powder	?4％

?D1，D7	Sucrose	?4％
			?D2，D8	Turanose	?4％
?D3，D9	Pyruvic acid methyl fat	?4％
			?D4，D10	The monomethyl succsinic acid	?4％
?D5，D11	The cis equisetic acid	?2％
			?D6，D12	Two hydration trisodium citrates	?2％

?E1，E7	Sodium formate	?2％
			?E2，E8	2%DL-Alpha-hydroxy Sodium propanecarboxylate	?2％
?E3，E9	DL-beta-hydroxy-butanoic acid sodium	?2％
			?E4，E10	Somsanit	?2％
?E5，E11	The 2%P-hydroxyl phenylacetic acid	?2％
			?E6，E12	D, the L-Sodium.alpha.-hydroxypropionate	?2％

?F1，F7	Propanedioic acid	?2％
			?F2，F8	The acid of D-granulated sugar	?2％
?F3，F9	The amino hydrochloride of L-L-Ala	?4％

?F4，F10	The L-L-Ala	?2％
			?F5，F11	The L-alanyl-glycine	?2％
?F6，F12	Altheine acid	?4％

?G1，G7	The L-Sodium Glutamate	?4％
			?G2，G8	The L-L-Histidine hydrochloride	?2％
?G3，G9	Hydroxyl-L-proline(Pro)	?2％
			?G4，G10	The L-leucine	?3％
?G5，G11	The L-Pyrrolidonecarboxylic acid	?2％
			?G6，G12	The D-Serine	?2％

?H1，H7	γ-An Jidingsuan	?2％
			?H2，H8	Inosine	?2％
?H3，H9	Thymine deoxyriboside	?2％
			?H4，H10	The putrescine dihydrochloride	?2％
?H5，H11	D, L-α-Phosphoric acid glycerol esters disodium	?2％
			?H6，H12	Alpha-D-glucose-1-phosphoric acid salt	?2％

2, configuration broth culture

(1), the proportional mixing of following each material configuration GN/MT (gram-negative bacteria salt storage liquid) (weight % concentration)

The material title	Proportion
		Pure water [H 2O]	61.07325％
Two hypophosphite monohydrate sodium dihydrogen [NaH 2PO 4-2H 2O]	10.6％
		Sodium phosphate, dibasic [Na 2HPO 4]	18.7％
Repone K [KCl]	7.455％
		Ammonium sulfate [(NH 4) 2SO 4]	2.114％
Sodium pyrophosphate decahydrate [Na 4P 2O 7-10H 2O]	0.01775％

(2), mix storage liquid by following material configuration concentration requirement configuration GN (gram-negative bacteria): (g/ml)

The material title	Concentration
		The mixed reagent of GN adds water configuration GN and mixes storage liquid	0.737％

(3), require to be configured to down the aqueous solution of several kinds of materials according to following configuration concentration: (g/ml)

The material title	Concentration
		0.5mM folic acid	0.022％
Viola crystallina	0.8％-1.1％
		Proteolytic enzyme	10-12％
Yeast extract paste	5％

(4), the mixed by following each material disposes broth culture: (v/v)

The material title	Proportion
		Pure water	65.8％
GN/MT	22.4％
		The GN mixed solution	2.24％

0.5mM the folic acid aqueous solution	0.89％
		The aqueous solution of protease of 10%-12%	1.49％
The 5% yeast extract paste aqueous solution	0.1％
		The Viola crystallina aqueous solution of 0.8%-1.1%	7.17％

3, packing

(1) quantitative requirement of producing in batches according to lath installs to the reaction substrate stoste branch that suitable step 1 disposes in 96 test tubes.

(2) quantitative requirement of producing in batches according to lath joins the broth culture that suitable step 2 disposes in the test tube of (1).

4, application of sample packing lath

Use to divide assembling system in the corresponding position in 96 holes to specified evaluation lath application of sample, every hole adds 50ul;

5, lath is dry

Above-mentioned lath Air drying 16-18 hour;

6, packing

7, lath storage

Since 47 carbon sources on the identification plate of the present invention and nitrogenous source make up appraisable gram-negative bacteria kind have 300 surplus kind; Can identify multiple different types of bacteriums such as negative enterobacteriaceae, vibrionaceae, Rhodopseudomonas, acinetobacter, narrow food zygosaccharomyces, negative severe bacteria; And need not replenish experiment and just can draw qualification result, simple to operate, the result is accurate; Thereby, bigger clinical value is arranged.

Embodiment:

Identification plate for gram negative aerobic bacteria, the technological process of production of producing 375 products in batches is following:

1, configuration identification plate reaction substrate stoste

The position, hole	Medicine name	This batch of pure water aequum (ml)	This batch of medicine aequum (g)
				A1，A7	Water	17	17
A2，A8	Tween 80	17	1.02
				A3，A9	N-ethanoyl-D-galactosamine	17	0.34
A4，A10	The L-pectinose	17	0.68
				A5，A11	The D-arabitol	17	0.68
A6，A12	The D-cellobiose	17	0.68
				B1，B7	D-fructose	17	1.36
B2，B8	The D-semi-lactosi	17	0.68
				B3，B9	Alpha-D-glucose	17	1.7

B4，B10	Glycerine	17	0.34
				B5，B11	α-D-lactose	17	0.68
B6，B12	SANMALT-S	17	0.68
				C1，C7	D-N.F,USP MANNITOL	17	1.36
C2，C8	The D-melibiose	17	0.68
				C3，C9	Beta-methyl-D-glycoside	17	0.68
C4，C10	The D-psicose	17	0.68
				C5，C11	The D-raffinose-	17	0.68
C6，C12	The D-Sorbitol Powder	17	0.68
				D1，D7	Sucrose	17	0.68
D2，D8	Turanose	17	0.68
				D3，D9	Pyruvic acid methyl fat	17	0.68
D4，D10	The monomethyl succsinic acid	17	0.68
				D5，D11	The cis equisetic acid	17	0.34
D6，D12	Two hydration trisodium citrates	17	0.34
				E1，E7	Sodium formate	17	0.34
E2，E8	2%DL-Alpha-hydroxy Sodium propanecarboxylate	17	0.34
				E3，E9	DL-beta-hydroxy-butanoic acid sodium	17	0.34
E4，E10	Somsanit	17	0.34
				E5，E11	The 2%P-hydroxyl phenylacetic acid	17	0.34
E6，E12	D, the L-Sodium.alpha.-hydroxypropionate	17	0.34
				F1，F7	Propanedioic acid	17	0.34
F2，F8	The acid of D-granulated sugar	17	0.34
				F3，F9	The amino hydrochloride of L-L-Ala	17	0.68
F4，F10	The L-L-Ala	17	0.34
				F5，F11	The L-alanyl-glycine	17	0.34
F6，F12	Altheine acid	17	0.68
				G1，G7	The L-Sodium Glutamate	17	0.68
G2，G8	The L-L-Histidine hydrochloride	17	0.34
				G3，G9	Hydroxyl-L-proline(Pro)	17	0.34
G4，G10	The L-leucine	17	0.51
				G5，G11	The L-Pyrrolidonecarboxylic acid	17	0.34
G6，G12	The D-Serine	17	0.34
				H1，H7	γ-An Jidingsuan	17	0.34
H2，H8	Inosine	17	0.34
				H3，H9	Thymine deoxyriboside	17	0.34
H4，H10	The putrescine dihydrochloride	17	0.34
				H5，H11	D, L-α-Phosphoric acid glycerol esters disodium	17	0.34
H6，H12	Alpha-D-glucose-1-phosphoric acid salt	17	0.34

2, configuration broth culture

(1), with following each material mixed configuration GN/MT salt storage liquid

The material title

This batch of medicine aequum

Pure water [H 2O]	Constant volume 400ml
		Two hypophosphite monohydrate sodium dihydrogen [NaH 2PO 4-2H 2O]	42.4g
Sodium phosphate, dibasic [Na 2HPO 4]	74.96g
		Repone K [KCl]	29.82g
Ammonium sulfate [(NH 4) 2SO 4]	8.456g
		Sodium pyrophosphate decahydrate [Na 4P 2O 7-10H 2O]	0.071g

(2), require configuration GN to mix storage liquid according to configuration concentration

The material title	This batch of pure water aequum (ml)	This batch of medicine aequum (g)
			The GN mix reagent	25	0.18425

(3), require to be configured to down several kinds of aqueous solution according to configuration concentration

The material title	This batch of pure water aequum (ml)	This batch of medicine aequum (g)
			0.5mM folic acid	10	0.0022
Viola crystallina	75	0.6-0.825
			Proteolytic enzyme	17.5	1.75-2.1
Yeast extract paste	5	0.25

(4) with following each material mixed configuration broth culture

The material title	This batch of medicine aequum
		Pure water	643.75ml
GN/MT	219.5ml
		GN mixes storage liquid	21.95ml
0.5mM the folic acid aqueous solution	8.75ml
		Aqueous solution of protease	14.625ml
The yeast extract paste aqueous solution	1.025ml
		The Viola crystallina aqueous solution	70.25ml

3, packing

(1) the reaction substrate stoste branch with configuration installs in corresponding 96 test tubes, and every pipe adds 6.675ml.

(2) broth culture with configuration joins in 96 test tubes, and every pipe adds 8.95ml.

4, application of sample packing lath

96 cuvette cartridges that configure are being added on the model machine, using to add model machine to specified evaluation lath application of sample, every hole adds 50ul.(to move the branch assembling system earlier before this; Whole procedure approximately needs 3 hours).

5, lath is dry

The lath that application of sample is good is put into drying room and is carried out Air drying, and lath can drying finish after 24 hours.

6, lath packing

The lath that drying is good is delivered to make-up room from drying room through pass-through and is packed.

7, lath storage

The lath that packing finishes is put into 2-8 ℃ of refrigerator and is preserved.

Claims (5)

1. identification plate for gram negative aerobic bacteria is characterized in that this identification plate is made up of reaction substrate stoste and broth culture, and position and the concentration of each composition thereof of said reaction substrate stoste on identification plate is: g/ml

Said broth culture is made up of following ingredients: v/v

2. according to the said a kind of identification plate for gram negative aerobic bacteria of claim 1, it is characterized in that the gram-negative bacteria salt storage liquid in the said broth culture is made up of following ingredients: weight % concentration

3. according to the said a kind of identification plate for gram negative aerobic bacteria of claim 1, it is characterized in that gram-negative bacteria in the said broth culture mixes gram-negative bacteria mix reagent that storage liquid is made up of following ingredients and adds water and dispose and form: weight % concentration

4. according to the said a kind of identification plate for gram negative aerobic bacteria of claim 1, it is characterized in that the substrate of said identification plate for gram negative aerobic bacteria and the position on identification plate thereof are:

5. the preparation method of an identification plate for gram negative aerobic bacteria is characterized in that this method comprises the following steps:

(1) configuration identification plate reaction substrate stoste

Require the stoste of each reaction substrate of configuration: g/ml according to following configuration concentration

(2) configuration broth culture

1. the proportional mixing of following each material configuration gram-negative bacteria salt is store liquid: weight % concentration

2. mix storage liquid g/ml by following material configuration concentration requirement configuration gram-negative bacteria

Material title concentration

The mixed reagent of gram-negative bacteria adds water configuration gram-negative bacteria to be mixed

0.737％

Close storage liquid

3. require to be configured to down several solns: g/ml according to following configuration concentration

4. the mixed by following each material disposes broth culture v/v

(3) packing

1. the quantitative requirement of producing in batches according to lath installs to the reaction substrate stoste branch that suitable step 1 disposes in 96 test tubes;

2. the quantitative requirement of producing in batches according to lath joins the broth culture that suitable step 2 disposes in the test tube 1.;

(4) application of sample packing lath

Use to divide assembling system in the corresponding position in 96 holes to specified evaluation lath application of sample, every hole adds 50ul;

(5) lath is dry

Above-mentioned lath Air drying 16-18 hour;

(6) packing;

(7) lath storage.