Novel Phage Amplified Multichannel Series Piezoelectric Quartz

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Novel Phage Amplified Multichannel Series Piezoelectric Quartz Crystal Sensor for Rapid and Sensitive Detection of Mycobacterium tuberculosis Xianwen Mi,†,‡ Fengjiao He,*,† Meiyu Xiang,† Yan Lian,† and Songlin Yi§ †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China, 410082 ‡ Huaihua Medical College, Huaihua, Hunan Province, China, 418000 § Hunan Institute of Tuberculosis Control, Changsha, China, 410006 ABSTRACT: The key factors that control the spread and mortality rate of tuberculosis (TB) are rapid detection and diagnosis. However, the current detection of Mycobacterium tuberculosis (M. tuberculosis) cannot meet the recommended requirements for clinical diagnosis in turnaround time. In this paper, the feature of phage D29 that infects M. tuberculosis and Mycobacterium smegmatis (M. smegmatis) was combined with the sensitivity of multichannel series piezoelectric quartz crystal sensor (MSPQC) to detect M. tuberculosis. The phage D29 played a role of inhibiting the growth of M. tuberculosis and M. smegmatis. M. tuberculosis is used to protect phage D29 from being killed by ferrous ammonium sulfate (FAS) and carries phage D29 into the detection medium containing M. smegmatis. The action of M. smegmatis indicated the existence state of phage D29 in the detection medium. The growth curve of M. smegmatis obtained by MSPQC indicated the state of the growth of M. tuberculosis. Therefore, M. tuberculosis in the sample could be rapidly detected by evaluating the extent of inhibiting the growth of M. smegmatis compared with the normal growth of M. smegmatis. The detection of M. tuberculosis was transformed into the detection of M. smegmatis, which is more rapid and sensitive than that of M. tuberculosis. For 102 cfu/mL of M. tuberculosis in clinical sample, the turnaround time was less than 30 h. Although statistical analysis showed that no significant difference existed between the results of the proposed method here and the BACTEC960 MGIT method in clinical M. tuberculosis detection, the phage amplified MSPQC (PA MSPQC) method presented here was faster and more economical.

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staining microscopy and light-emitting diode (LED) fluorescence microscopy for diagnosis4 and further analyzed the effect of the method for the collection of sample on the detected results.5 The smear microscopy possessed an advantage of rapid diagnosis, but the sensitivity was only 50%, which is not adaptable to clinical diagnostic criteria. Bacterial culture technique included Lowenstein-Jensen (L-J) slants, thin-layer agar, and the BACTEC 460TB and BACTEC MGIT 960 systems. Compared with the turnaround time of L-J slant culture (30 days),9 although the BACTEC system has shortened the turnaround time to 10 days, it could not meet the requirements of clinical diagnosis for the turnaround time. However, it is difficult to be popularized due to its high equipment cost and reagents consumption. Serological diagnosis, including enzyme-linked immunosorbent assay (ELISA) 1 1 , 1 2 and dot immunogold filtration assay (DIGFA),13−15 is unable to meet the demands for sensitivity and specificity because the type and quantity of immune

ccording to the data published by the World Health Organization, there are about 2 billion people infected with M. tuberculosis; in 20 million sick people, 3 million people die from it every year.1 People suffering from and ultimately dying of M. tuberculosis are mainly distributed in developing countries, and most of them are of the age of 20 to 50 years. For the appearance of resistant strains of M. tuberculosis, the spread of AIDS, and people migration, the control of tuberculosis (TB) has become a worldwide challenge for public health. Drug susceptibility tests and rational administration are effective ways to prevent the appearance of drug resistance strains and to control the propagation of pathogenic M. tuberculosis. Therefore, rapid detection of M. tuberculosis and diagnosis of TB patients is one of the key factors to control TB.2 Nowadays, a great deal of methods, such as smear microscopy,3−5 bacterial cultivation assay,6−10 serological diagnosis,11−17 molecular biology diagnostic techniques,18−25 and multichannel series piezoelectric quartz crystal sensor (MSPQC) methods26−31 are developed to detect M. tuberculosis. In the developed process for the smear technique, Farnia developed a new way by treating sputum samples with chitin.3 Luis described the advantage and disadvantage of Ziehl-Neelsen © 2011 American Chemical Society

Received: August 8, 2011 Accepted: December 5, 2011 Published: December 5, 2011 939

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ferrous ammonium sulfate (FAS), and phage inside M. tuberculosis was still alive.35 Then, the sample solution mentioned above was added into the detection medium containing M. smegmatis; M. smegmatis in the detection medium was infected with phage D29 released from M. tuberculosis, and the growth of M. smegmatis was restricted. Finally, the response curve of M. smegmatis growth inhibition was acquired by the MSPQC system. Compared with normal growth of M. smegmatis, the state of M. smegmatis growth inhibition and the existence of M. tuberculosis in sample could be evaluated. The turnaround time shortened to 30 h. All in all, phage amplified MSPQC (PA MSPQC) for the detection of M. tuberculosis has the advantage of being rapid, specific, safe, and cheap.

expression of M. tuberculosis varied with the background of the immune system and disease course of the patient.16,17 As for molecular technologies, a lot of research on molecular techniques in TB laboratory detection was completed to shorten the turnaround time,18 improve the detection sensitivity,19−21 analyze the mutants of resistant strains,22,23 and type the substrains of clinical sample.24,25 In tandem with the development of molecular technology, it will become the most powerful tool for the detection, identification, and drug susceptibility testing of M. tuberculosis. However, the application of molecular diagnosis technology is restricted because of the harsh experimental conditions and experimental costs in the developing country. Therefore, it is necessary to develop a new detection method for the detection of M. tuberculosis with characteristics of being rapid, sensitive, specific, economical, and safe. Recently, piezoelectric quartz crystal sensor technology has been proposed for rapid detection of microorganisms based on the changes of electrical parameters in the medium during the growth process.26−29 The turnaround time for detecting M. tuberculosis has been cut short to 7 days.30,31 However, it is difficult to further shorten the turnaround time due to its generation time and the problem of lacking specificity in electrochemical methods. Thus, a desirable new method to overcome these defects was developed. Phage D29 can not only infect M. tuberculosis and M. smegmatis32 but also generate the offspring much more than that of the binary of fission of bacteria.33 McNerney developed the FASTPlaque test for rapid detection and assessment of drug susceptibility for M. tuberculosis from clinical samples34 by improving phage detection technology.35 Rardarov detected M. tuberculosis and assessed drug susceptibility of M. tuberculosis in clinical samples using luciferase reporter phage in comparison with the Mycobacterium Growth Indicator Tube system.36 The results of these investigations showed that the sensitivity of FASTPlaque test was up to 60%, which was better than that of smear microscopy.37 Further, Marei discovered that the sensitivity of FASTPlaque in the urine sample was almost 100%, much better than that in sputum sample.38 However, the investigation results obtained from the National Laboratory of Zambia by McNerney showed that the performance of FASTPlaque test in the developing countries had no advantage compared with smear microscopy. 39 The results of FASTPlaque test were observed on agar plate. A single or a colony of mycobacterium infected with phage D29 can only form a plaque on the solid agar plate. Because M. smegmatis was cultivated on solid agar plate, the characteristics phage D29 possessed of biomagnifications in vivo of M. smegmatis and the sensitivity of FASTPlaque test were restricted. On the contrary, because the liquid medium is substituted for the solid agar plate, it is an effective way that the phage D29 number can be amplified at geometric series in liquid medium containing M. smegmatis to improve detection sensitivity of the phage method. In this paper, therefore, the solid agar plate was replaced by liquid medium to give full play of phage amplification. The characteristic of M. tuberculosis and M. smegmatis infected with phage D29 was combined with the sensitivity of MSPQC to the electrical parameter of the medium for detecting M. tuberculosis. Thus, the detection of M. smegmatis was substituted for M. tuberculosis using phage D29 as a bridge to connect M. smegmatis with M. tuberculosis. The detection process was described as follows: First, M. tuberculosis was infected with phage D29; phage D29 outside M. tuberculosis was killed by

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EXPERIMENTAL SECTION Bacteria Isolates and Bacteriophage. M. smegmatis (ATCC607, dry powder) and phage D29 (dry powder) were purchased from Institute of Microbiology Chinese Academy of Sciences. Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and M. tuberculosis (H37Ra) were bought from National Institute for the Control of Pharmaceutical and Biological Production. M. smegmatis was inoculated in M7H9 medium, collected from logarithmic growth phase and then stored at −70 °C. Phage D29 cultured in M7H9 medium was preserved in a refrigerator at 4 °C. M. tuberclosis, E. coli, S. aureus, and P. aeruginosas were saved in a refrigerator at 4 °C. Reagents and Medium. The detection medium of ammonium sulfate (0.05 g), sodium hydrogen phosphate (0.25 g), potassium dihydrogen phosphate (0.1 g), magnesium sulfate (0.005 g), zinc sulfate (0.001 g), copper sulfate (0.001 g), sodium glutamate (0.01 g), sodium citrate (0.01 g), pyridoxine hydrochloride (0.001 g), biotin (0.0005 g), and ferric ammonium citrate (0.04 g) was dissolved in 1 L of distilled water, and then, it was adjusted to pH 7.2 with ammonia solution. The medium was separated into 100 mL bottles and autoclaved at 121 °C for 15 min. Five milliliters of 10% oleic acid−albumin−dextrose−catalase (OADC)7 was added into each of the bottles. Finally, 1.0 mL of 0.1 mol/L calcium chloride was added into the repackaged broth before use. For the sample treatment solution, first, 4% sodium hydroxide solution and 2.9% citrate solution were mixed at a ratio of 1:1 (v/v). The mixed solution was autoclaved at 121 °C for 10 min. N-Acetyl-L-cysteine (NALC; 0.5 g) was added into the mixed solution (0.5% w/v). For the M. smegmatis solution (108 cfu/mL), first, M. smegmatis revived at ambient temperature was inoculated on L-J slants and incubated at 37 °C. After 40 h, pure lawns were scraped from slants by sterile ring vaccination and put into sterile porcelain mortar. Bacterial suspension was made by grinding and mixing with detection medium in the porcelain mortar. Finally, 108 cfu/mL of M. smegmatis suspension was made for use according to McFarland’s turbidity. For phage D29 solution (108 pfu/mL), different titer phage D29 solutions were prepared by 10-fold serial dilution. For the positive control solution, 1.0 mL of 106 cfu/mL of H37Ra solution was used. The mixtures of E. coli (105 cfu/mL), S. aureus (105 cfu/mL), and P. aeruginosa (105 cfu/mL) were used as negative control. For phage D29 control solution, 1.0 mL of 106 pfu/mL of phage D29 solution was used. Phosphate buffer solution (PBS, 0.067 mol/L), 0.1 mol/L calcium chloride 940

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where ΔF is the frequency shift (F0 is the fundamental frequency of the piezoelectric crystal); C0 and Cp are static capacitance and motional capacitance of the crystal, respectively. G and Cs are the conductance and capacitance of solution, respectively. G = κχ, and Cs = κε + Cp, (K is the cell constant; X is the specific conductivity; ε is the solution permittivity; Cp is the parasitic capacitance between the leading wires of the electrodes). Y is a parameter related to phase shift of the oscillator. Equation 1 could be simplified as eq 2 under the experimental condition

solution, 4% ferrous ammonium sulfate (FAS) solution, and McFarland’s turbidity tubes were prepared for use. For the sample solution, 2.0 mL of sputum sample solution was added into 15 mL centrifuge tubes containing 4.0 mL of sample treatment solution with a screw cap. The sample was dispersed by a vortex oscillator and stood at room temperature for 15 min. Sterile 0.067 mol/L phosphate buffer (pH 6.8, 9.0 mL) was added into the tube and mixed thoroughly, and it was centrifuged at 4000 rpm for 15 min; second, the supernatant was discarded, and the sediment was washed with 15 mL of sterile PBS buffer and M7H9 medium, respectively. One milliliter of detection medium and 0.1 mL of phage D29 solution (108 pfu/mL) were added to the tube containing sediment and incubated for 1 h at 37 °C. Finally, 0.1 mL of 4% FAS was added to inactivate free phage D29 for 5 min. The suspension was diluted to 10 mL, and the sample solution was ready for detection. All chemicals used here were analytical pure grade. Detection System. The MSPQC system was custom-made for our lab.26 The block diagram of the MSPQC system for rapidly detecting a bacterium is shown in Figure 1.29 This

(2) ΔF = − k ΔG k is considered to be a constant. This is the basic principle of MSPQC analysis. BACTECTM MGIT 960 system was produced by Becton & Dickinson Corporation. Detection Procedure. Sample treatment solution (0.5 mL) was added into the detection cell which contained 4.0 mL of detection medium and 0.5 mL of M. smegmatis suspension (108 cfu/mL). Positive control solution (0.5 mL), 0.5 mL of negative control solution, and phage D29 control solution were treated using the same process mentioned above. For comparison, 1.0 mL of sample treatment solution was inoculated in a growth indicator tube of MGIT 960. Frequency response curves were recorded by the MSPQC system. All positive results were timely confirmed by acid-fast smear. All experiments described here were operated in class II biological safety cabinets, and all experiment wastes and utensils were washed after autoclaving treatment.

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RESULTS AND DISCUSSION Construction of the PA MSPQC Sensor for Rapid Detection of M. tuberculosis. Growth Curves of M. smegmatis and M. tuberculosis Detected by the MSPQC Sensor. The growth response curves of M. smegmatis (curve a) and M. tuberculosis (curve b) in M7H9 medium detected by MSPQC were shown in Figure 2. The curve included rising

Figure 1. Block diagram of a multichannel series piezoelectric quartz crystal sensor system.

system consists of three major components. Part I is an array consisting of 32 sensors that produce the response signal to microbial growth. Marker 1 was detection cells, and marker 2 was the piezoelectric quartz crystal. Part II is a microprocessor system processing the sensor signal according to the illustrations; Part III is a data output system, which sets experimental parameters and observes the signal online by Elite software. This system is sensitive to the change of electrical parameters in solution;40 the value of frequency shift can be expressed as eq 1:

ΔF =

Figure 2. Growth response curve of M. smegmatis and M. tuberculosis in M7H9 medium: a, M. smegmatis; b, M. tuberculosis; c, medium control.

phase, turning point, and plateau phase. The rising phase indicated a period that M. smegmatis multiplied, the concentration of M. smegmatis was increased, and the value of frequency shift was rising up. The turning point in curves a and b demonstrated that the conductive ingredient was entirely consumed only in the presence of M. smegmatis in the detection medium or that all M. smegmatis was infected by phage D29

πF0 2Cq(4π 2F0 2Cs2Y + 4πF0CsG − YG2) [4π 2F0 2Cs(C0 + Cs) − 2πF0C0YG + G2]2 ·ΔG

(1) 941

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when M. smegmatis and phage D29 coexisted in the detection medium, and the frequency shift arrived at the maximum. Beyond the turning point, the frequency shift of the plateau phase does not change and it seemed to level off even when the time is up to 300 h. The reason is the fact that the growth of M. smegmatis stopped while there was no change of conductance in the solution. Therefore, a steady value was obtained. As described in Figure 2, the frequency shift obtained by MSPQC responded to M. smegmatis and M. tuberculosis. Compared with M. tuberculosis (curve b), the frequency shift of M. smegmatis (curve a) rose up sharply in a short time; the time of rising phase is only 30 h, after M. smegmatis was inoculated in M7H9 medium. It is noticed that the frequency shift is up to 400 Hz (turning point appeared). Yet, for M. tuberculosis, 300 Hz was obtained after 250 h in curve b. It suggested that the turnaround time of M. smegmatis is only 30 h, while the turnaround time of M. tuberculosis is prolonged to 250 h. The reason was that M. smegmatis is a fast growing mycobacterium and its generation time is only 4 h, while the generation time of M. tuberculosis is 18 h. Therefore, the turnaround time of the detection method was sharply shortened to 30 h. The response signal was generated due to sodium citrate and ferric ammonium citrate being utilized during the growth process of M. smegmatis.41,42 To explore the reason of the generation of signal, the blank experiments on the utilization of sodium citrate (5 × 10−4 mol/L) and ferric ammonium citrate (5 × 10−4 mol/L) by M. smegmatis were also performed. The results are depicted in Figure 3. The value in the detection

equal to that of dihydrogen phosphates. So, phosphate buffer could balance the change of conductance which resulted from the change of the concentration of H+ and OH−. On the basis of the analysis of signal intensity of curves a, b, and c, among their components in the detection medium, a conclusion was drawn that the response signal of the MSPQC system was caused by the utilization of conductive ingredients, such as citrate, ammonium, and ferric in the detection medium. Construction of the PA MSPQC Sensor for Rapidly Detecting M. tuberculosis. The treated sample was added to the detected cell. The scheme for the detection principle is shown in Figure 4. Phage D29 was added to sample solution

Figure 4. Flowchart of PA MSPQC for M. tuberculosis detection.

and incubated for 1 h. M. tuberculosis in sample solution was infected by phage D29; the FAS solution was added into sample solution to inactivate free phage D29 outside M. tuberculosis, and then, sample solution was 10-fold diluted to eliminate the role of FAS. This dilute solution was transferred to the detection cell containing M. smegmatis in the detection medium. When there was no M. tuberculosis in sample solution, no phage D29 would be carried into the detection medium. The frequency curve was recorded by MSPQC. Effect of Different Concentrations of Phage D29 on the Growth of M. smegmatis Detected by MSPQC. The effect of different concentrations of phage D29 on the propagation of M. smegmatis was shown in Figure 5. The reproduction of M. smegmatis was not repressed without adding phage D29 (curve a) and almost completely inhibited when concentration of phage D29 is 104 pfu/mL (curve f). Compared with the proliferation rate of M. smegmatis in the detection medium, phage D29 increased faster. As long as phage D29 exists in the detected system, the growth of M. smegmatis will be completely inhibited. It was indicated that the inhibition of M. smegmatis growth depended on the concentration of phage D29 in detection medium. The lower was the concentration of phage D29, the longer the time would be to arrive at turning point time (T) and the higher the frequency shift would be observed. Turning point of the curve showed that the concentration of phage D29 was equal to that of M. smegmatis after a period time of cultivation. The response curve of M. smegmatis reached a plateau phase when the growth of M. smegmatis was entirely inhibited.

Figure 3. Response curves of M. smegmatis (107 cfu/mL) in detection medium with specific ingredients: a, 5 × 10−4 mol/L ferric ammonium citrate; b, 5 × 10−4 mol/L sodium citrate; c, phosphate control.

medium with ferric ammonium citrate (curve a) was larger than that with sodium citrate in the detection medium (curve b). No obvious frequency shift appeared in the phosphate control (curve c). The results indicated that the citrate could be specifically used as carbon source and ammonium as nitrogen sources and ferric ion was specifically adsorbed by M. smegmatis in curve a. Only citrate could be used as carbon source in curve b. Phosphate, as a buffer which could neutralize hydrogen ion generated in the process of glucose metabolism of M. smegmatis, had a significant role to maintain the stability of acidity environment for the growth of M. smegmatis in the detection medium. Hydrogen ions were neutralized by hydrogen phosphate and were transformed into dihydrogen phosphate. Molar conductivity of hydrogen phosphate was 942

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Figure 5. Efect of different concentrations of phage D29 on the growth of M. smegmatis: a, 0 pfu/mL; b,1 pfu/mL; c, 10 pfu/mL; d, 102 pfu/mL; e, 103 pfu/mL; f, 104 pfu/mL.

Figure 6. One step growth curve of phage D29 in M. smegmatis.

and cell cycle was 4 h.43 Under the experimental conditions, the concentration of M. smegmatis was set at 107 cfu/mL. Equation 5 could be simplified as eq 6. Therefore, the time (T) depended on the concentration of phage D29 (Cp).

When all M. smegmatis was infected by phage D29 and lost the ability to reproduce, the turning point appeared. On the basis of the importance of turning point in the frequency shift, it is necessary to construct a mathematical model to illustrate the relationship between the time of turning point and the concentration of phage D29. The turning point time (T) in the frequency shift curve depended on the initial concentration (Cp, pfu/mL), burst size (Qp), and average lytic cycle (tp, hour) of phage D29 and the initial concentration (Cm, cfu/mL), proliferating number (Qm), and cell cycle (tm, hour) of the M. smegmatis. The relationship among these parameters can be expressed by eq 3:

T = 15 − 2.7 logC p

It could be calculated that the time of turning point was 15 and 10 h by eq 6, respectively, when titer of phage D29 was 1 pfu/mL and 100 pfu/mL after inoculation. Considering the adaptation period of M. smegmatis in the detection medium is about 3 h, the calculated results were consistent with the experimental results. As stated previously, it can be deduced that the plateau of frequency shift curve was attributed to the inhibition of M. smegmatis by phage D29, and the existence state of phage D29 could be evaluated according to the extent of inhibition for M. smegmatis growth. According to the results of experiments, we can depict the relationship between logarithm of phage D29 number and time of turning point in Figure 7. From the curve, we can draw that

C p × Q pT/t p = C m × Q m T/t m − C p × (Q p0 + Q p1 + ··· + Q p((T/t p) − 1) + Q pT/t p)

(6)

(3)

The function of Cp × QpT/tp indicated that the concentration of phage D29 was increasing with the time in a geometric progression, while the expression Cm × QmT/tm showed that the concentration of M. smegmatis was increasing with the time in a geometric progression in the absence of phage D29. The function Cp × (Qp0 + Qp1 + ··· + Qp((T/tp) − 1) + Qp(T/tp)) meant the sum concentration of M. smegmatis infected by the generation of phage D29. After logarithmic processing, eq 3 was transformed into eq 4

T logC p + log(2Q p) + · logQ p tp T = logC m + ·logQ m tm

(4) Figure 7. Relationship between the logarithm of phage number and time of turning point.

The time (T) when frequency shift arrived at the maximum can be expressed by eq 5

T=

the linear range of standard curve was narrow only from 1pfu/ mL to 102 pfu/mL and the time of turning point deviated from the standard curve because the adsorption of phage D29 to M. smegmatis was uncertain at higher concentration. Effect of Different Concentrations of M. smegmatis on the Frequency Shift Curves. The frequency shift curves of M. smegmatis at different concentrations in detection medium are shown in Figure 8. It is indicated that the time of turning

t m·t p·[logC m − logC p − log(2Q p)] t m·logQ p − t p·logQ m

(5)

One step growth curve of phage D29 in M. smegmatis was shown in Figure 6. The latency is 100 min; burst size (Qp) is 15, and the average lytic cycle (tp) is 150 min. The reproduction of M. smegmatis with binary fission (Qm = 2) 943

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Figure 9. Effect of Ca2+ on the interaction between M. smegmatis (107 cfu/mL) and phage D29: a, 0.002 mol/L calcium ion with no phage D29; b, phage D29 (105 pfu/mL) without calcium ion; c, phage D29 (105 pfu/mL) with 0.002 mol/L calcium ion.

Figure 8. Response curves of different concentration of M. smegmatis: a, 108 cfu/mL; b, 107 cfu/mL; c, 106 cfu/mL; d, 105 cfu/mL; e, 104 cfu/mL.

the role of increasing productive infection and adsorption rate in the process of phage interaction with M. smegmatis. The infection efficiency of phage D29 to M. smegmatis was affected by the adsorption and abortive infection, followed by the process of infection of phage D29 to M. smegmatis. Abortive infection was a prominent feature following adsorption of phage D29 at the surface of M. smegmatis. Of all the cations added to the ordinary nutrient broth, only calcium ion could increase the rate of adsorption and productive infection. Calcium ions could chelate with DNA and the enzyme located on the surface of cell, suitably orienting both enzyme and substrate, or activate an enzyme related to helping DNA to get through the cell wall barrier. In Sellers’ experiments, supplementing the medium with calcium not only increased adsorption rate of phage D29 to M. smegmatis from 21% to 66% but also reduced its abortive infection rate from 62% to 20%.44 Typical Response Curve of M. tuberculosis Detected by PA MSPQC. The typical response curves for M. tuberculosis detected by the proposed method were shown in Figure 10.

point appearance was relative with the concentration of M. smegmatis. The higher the concentration of M. smegmatis, the earlier the turning point appeared. The time of turning point indicated the complete absorption of conductance ingredient by M. smegmatis on the condition that only M. smegmatis existed in the detection cell. The turnaround time depended on the time of turning point of M. smegmatis. When M. semgmatis coexisted with phage D29, the relationship of turning point time (T) and the concentration of M. smegmatis and phage D29 can be expressed as eq 7 transformed from eq 5.

C T = 2.7 log m − 4 Cp

(7) 8

When the concentration of M. smegmatis was up to 10 cfu/mL and the concentration of phage D29 was 1 pfu/mL, the difference between the values of turning point of frequency shift curve in the M.smegmatis-phage D29 system and M. smegmatis system was not enough to judge the positive and negative. The time of turning point (curve e) was too long when the concentration of M. smegmatis was at 104 cfu/mL. Therefore, the optimum concentration of M. smegmatis in the detection system was 107 cfu/mL. Effect of Ca2+ on the Interaction between Phage D29 and M. smegmatis. The effect of calcium ion on the interaction of M. smegmatis and phage D29 was presented in Figure 9. In curve a, the value of growth signal of M. smegmatis at the concentration of 107 cfu/mL in the detection medium with 0.002 mol/L calcium chloride arrived at 400 Hz after inoculation for 25 h; curve b described the signal for 107 cfu/ mL of M. smegmatis interacting with 105 pfu/mLof phage D29 without calcium ion in the detection medium. In curve b, the signal reached a plateau when frequency shift value arrived at the maximum 150 Hz after inoculation for 20 h in the absence of calcium ion. While there were sufficient phage D29 in the detection medium, it took 20 h to completely inhibit the growth of M. smegmatis due to the lower absorption efficiency and higher abortive infection rate of phage D29 to M. smegmatis. Curve c depicted the growth signal of M. smegmatis at a concentration of 107 cfu/mL interaction with 105 pfu/mL phage D29 in the detection medium containing 0.002 mol/L calcium chloride. In curve c, although the concentration of M. smegmatis and phage D29 were the same as in curve b, there was no significant frequency shift observed. Calcium ion plays

Figure 10. Response curves of positive and negative sample in M7H9 medium: a, negative sample; b, 102 cfu/mL of M. tuberculosis; c, 107 cfu/mL of M. tuberculosis.

Curve a described the frequency shift curve of negative sample without M. tuberculosis. All phage D29 added to the sample treatment solution were inactivated by FAS, and there was no activated D29 transported to the detection medium. The curve a was obtained from the steps a′ to d′ in Figure 4. The curve b and c depicted the frequency shift curves of positive samples with M. tuberculosis. The process of curves b and c were corresponding to the steps of the detection flowchart from a to 944

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d in Figure 4. First, the phage D29 adsorbed on to the surface of M. tuberculosis and injected its DNA in vivo. The phage D29 in vivo of M. tuberculosis was protected to avoid the killing by FAS. The sample solution containing DNA of phage D29 was transferred into detection medium; finally, the growth of M. smegmatis was entirely inhibited by the phage released from M. tuberculosis. In the curve b, the concentration of M. tuberculosis was 100 cfu/mL. Identification of the Criteria for Positive Sample and the Detection Limit of the Proposed Method. Negative control sample was prepared according to the method described in Reagents and Medium, and parallel experiments of negative control were performed according to the method described in Detection Procedure. The value of frequency shifts of 15 negative samples was 291, 314, 330, 334, 340, 345 342, 348, 356, 359, 363, 369, 370, 388, and 422 (Hz), respectively. Its average and standard deviation was 351 and 32 (Hz), respectively. The confidence interval of 99% frequency shift value in the negative sample (x̅ ± 3Sb) is from 255 to 447 (Hz). The sample whose frequency shift was more than 255 was judged to be negative, on the contrary, to be positive. Different concentrations for positive samples, 10, 102, 103, and 104 cfu/mL, were prepared according to the described method in Reagents and Medium. The average values of frequency shift curve of five parallel sample concentrations at 10, 102, 103, and 104 cfu/mL were 298, 142, 70, and 30 Hz, respectively. Therefore, according to the criteria for the positive-negative assessment, the detection limit of the proposed method here was 100 cfu/mL. Detection of Clinical Samples. Treatment of Clinical Samples. Clinical samples may contain M. tuberculosis as well as Staphylococcus, Streptococcus, Meningitis bacteria, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and other different strains of bacteria. Contamination of these microorganisms would lead to false negative results. Thus, the correct treatment process of infected sample is crucial to detect M. tuberculosis accurately. High lipid content of cell wall of M. tuberculosis made them more tolerant to the killing of acid and alkaline solutions than other microorganisms. Four percent sodium hydroxide can kill other microorganisms without affecting the growth and reproduction of M. tuberculosis. Therefore, clinical samples can be treated with the method described in Detection Procedure. Two simulating positive samples (sputum of normal people + E. coli. + S. aureus + P. aeruginosa (105 cfu/mL) + M. tuberculosis H37Ra (106 cfu/mL) and two simulating negative samples (sputum of normal people + E. coli. + S. aureus + P. aeruginosa (105 cfu/mL) treated with protocol according to the method in Detection Procedure were prepared for the next experiments. A panel containing a positive and negative sample was incubated in the PA MSPQC system. The others were diluted to 10 mL directly and incubated in the PA MSPQC system for comparison. The treated samples can be detected by our system, while the untreated sample cannot be detected in Figure 11. The results of treated samples (curve c and curve d) have significant difference between the positive and negative samples. The results of untreated sample have no significant difference between positive and negative samples (curve a and curve b) because of the growth of contaminated microorganisms. It is necessary to treat the sample before detection. Sensitivity and Specificity of Clinical Sample Detection. The clinical sample (83), collected from Hunan Institute of Tuberculosis Control (Changsha China), was detected for M.

Figure 11. Frequency shift curves of treated sample and untreated sample: a, untreated negative sample; b, untreated positive sample; c, treated negative sample; d, treated positive sample.

tuberculosis by PA MSPQC, L-J slant culture, and BACTEC 960, respectively, adopting a double-blind trial. The positive results obtained by the BACTEC 960 MGIT system were confirmed with acid-fast staining. The sensitivity and specificity of the PA MSPQC method were evaluated by culture method, which was 89% (23/27) and 95% (53/56), respectively, shown in Table 1. Compared with sensitivity of FASTPlaque (60%), Table 1. Evaluation of PA MSPQC by Culture Method culture method PA MSPQC

+

−

total

+ − total

24 3 27

3 53 56

27 56 83

the sensitivity of the proposed method (89%) has been greatly improved by PA MSPQC to meet the requirements of clinical detection for M. tuberculosis. The results of the difference between the PA MSPQC and BACTEC 960 MIGT systems were summarized in Table 2. Table 2. Comparison of PA MSPQC Method with MGIT Method for Detecting 83 Clinical Samples BACTEC 960 MGIT PA MSPQC

+

−

total

+ − total

21 1 22

4 57 61

25 58 83

The detected results of 83 clinical samples obtained by BACTEC 960 MGIT and PA MSPQC were analyzed by a chi-square test for pair-count data. It was indicated that there was correlation between the results detected by the BACTEC 960 MGIT and PA MSPQC methods according to the value of the chi-square test (χ2 = 60 > χ20.05); according to the significant difference test (χ2 = 0.8 < χ20.05), there was no significant difference between two methods at P = 0.05 level. Although the specificity and sensitivity of PA MSPQC and BACTEC were high enough to meet clinical requirements, the turnaround time of PA MSPQC system was 30 h. This was far less than that of the MGIT960 system. Therefore, the PA MSPQC detection system possessed much significance for M. tuberculosis detection. 945

dx.doi.org/10.1021/ac2020728 | Anal. Chem. 2012, 84, 939−946

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CONCLUSIONS Utilizing the combination of recognition of phage D29 to M. tuberculosis and sensitive response of MSPQC to the growth of M. tuberculosis, a novel method for rapid, sensitive, and realtime detection of M. tuberculosis was developed. The turnaround time (30 h) was far less than that of BACTEC 960 MGIT. The detection limit was 100 cfu/mL. This was affected by phage infection efficiency. The assay feature of the proposed method could be improved by selecting more suitable phage and better sample handling. The developed method (PA MSPQC) offers a path for detection and drug susceptibility in the future.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 731 88272269. Fax: +86 731 88055818. E-mail: [email protected].

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ACKNOWLEDGMENTS This research work was supported by the National Natural Science Foundation of China (No.20875026) and Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).

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dx.doi.org/10.1021/ac2020728 | Anal. Chem. 2012, 84, 939−946